Cell having the potentiality of differentiation into cardiomyocytes

ABSTRACT

The present invention relates to methods for isolation, purification, expansion, and differentiation of cells having the potential to differentiate into cardiomyocytes. Furthermore, the present invention relates to methods for proliferating cells having the potential to differentiate into cardiomyocytes and for regulating their differentiation into cardiomyocytes using various cytokines and transcription factors. Moreover, the present invention relates to a method for obtaining surface antigens specific for cells having the potential to differentiate into cardiomyocytes, a method for obtaining genes encoding the surface antigens, a method for obtaining antibodies specific for the surface antigens, and a method for obtaining a protein and a gene controlling the proliferation of cells having the potential to differentiation into cardiomyocytes and their differentiation into cardiomyocytes. Also, the present invention relates to therapeutic agents for various heart diseases containing cells having the potential to differentiate into cardiomyocytes. Still furthermore, the present invention relates to a method for differentiating various cells and tissues such as neural cells, hepatocytes, adipocytes, skeletal muscle cells, vascular endothelial cells and osteoblasts, using cells having the potential to differentiate into cardiomyocytes.

TECHNICAL FIELD

[0001] The present invention relates to methods for isolation,purification, expansion, and differentiation of cells having thepotential to differentiate into cardiomyocytes. Furthermore, the presentinvention relates to methods for proliferating cells having thepotential to differentiate into cardiomyocytes and for regulating theirdifferentiation into cardiomyocytes using various cytokines andtranscription factors. Moreover, the present invention relates to amethod for obtaining surface antigens specific for cells having thepotential to differentiate into cardiomyocytes, a method for obtaininggenes encoding the surface antigens, a method for obtaining antibodiesspecific for the surface antigens, and a method for obtaining a proteinand a gene controlling the proliferation of cells having the potentialto differentiate into cardiomyocytes and their differentiation intocardiomyocytes. Also, the present invention relates to therapeuticagents for various heart diseases containing cells having the potentialto differentiate into cardiomyocytes.

BACKGROUND ART

[0002] Cardiomyocytes actively divide into daughter cells withspontaneous beating before birth. However, they lose the proliferativeactivity after birth and never acquire the division potentiality againunlike hepatocytes. Furthermore, unlike skeletal muscles, they does nothave undifferentiated precursor cells such as satellite cells.Therefore, when cardiomyocytes are necrotized by myocardial infarction,myocarditis, senility etc., hypertrophy of the remaining cardiomyocytesoccurs in vivo instead of cell division. Cardiac hypertrophy is aphysiological adaptation at the initial stage, but when coupled with thefibrosis of stroma by the growth of cardiac fibroblasts, it comes tolower the diastolic function and the systolic function of heart itself,leading to heart failure. Therapy so far developed for heart failurecaused by myocardial infarction, etc. is mainly symptomatic therapy, forexample, intensification of the cardiac systolic function, alleviationof the pressure overload and the volume load on heart using avasodilator drug, and decrease of blood flow using of a diuretic. On theother hand, heart transplantation is alternative therapy for severeheart failure, but is not generally adopted as a common treatmentbecause of problems such as shortage of heart donors, difficulty injudging cerebral death, immune rejection and a great rise in medicalcost. At present, heart diseases are the third cause of mortality inJapan (Annual Report on Health and Welfare, 1998), and thus success inregeneration of lost cardiomyocytes will lead to a great advance inmedical welfare.

[0003] As a cell line retaining the characteristics of cardiomyocytes,AT-1 cell line has been obtained from the atrial tumor of the transgenicmouse expressing SV40 promoter large T antigen under the control ofatrial natriuretic hormone promoter (Science, 239: 1029-1038 (1988)).However, this cell line forms tumors when transplanted in vivo and thusis inappropriate for cell transplantation. Under these circumstances,the following methods have been proposed for reconstructing myocardium.

[0004] The first method is conversion of cells other than cardiomyocytesinto cardiomyocytes, which has been proposed on the analogy of theconversion of fibroblasts into skeletal muscle cells by the introductionof MyoD. Although a successful result has been reported with P19 cellwhich is a murine embryonal carcinoma cell (Cell Struc. & Func., 21:101-110 (1996)), there has been no report on success withnon-carcinomatous cells.

[0005] The second method is restoration of proliferative activity tocardiomyocytes, which is based on the fact that beating cardiomyocytescan proliferate in the fetus. No successful example of this method hasbeen reported yet.

[0006] The third method is induction of cardiomyocytes fromundifferentiated stem cells. It has already been demonstrated thatcardiomyocytes can be differentiated from embryonic stem cells (EScells), but there still remain the problems of carcinoma formation andimmune rejection by embryonic stem cells transplanted into an adulttissue. (Nature Biotechnology, 17: 139-142 (1999)).

[0007] In order to practically utilize embryonic stem cells in medicaltreatments, it is essential to develop a technique for highly purifyingat least cardiomyocyte precursor cells or cardiomyocytes. As for theproblem of immune rejection, the possibility of solving the problem bythe cloning technique has been suggested, but it is difficult to applythis technique to general medical treatments because of its complicatedoperation.

[0008] It has also been proposed to transplant undifferentiatedcardiomyocyte precursor cells obtained from an aborted fetus, and it isknown that such cells effectively function as cardiomyocytes in anexperiment using animals (Science, 264: 98-101 (1994)). However, it isdifficult to obtain a large amount of cardiomyocyte precursor cells inthis method, and the method is hardly applicable to general medicaltreatments also from an ethical viewpoint.

[0009] It is known that there exist mesenchymal stem cells besideshematopoietic stem cells and vascular stem cells in adult bone marrowand that mesenchymal stem cells can be induced to differentiate intoosteocytes, chondrocytes, tendon cells, ligament cells, skeletal musclecells, adipocytes, stromal cells and hepatic oval cells (Science, 284:143-147 (1999); Science, 284: 1168-1170 (1999)). On the other hand, ithas been recently reported that the cells obtained from the bone marrowof an adult mouse can be induced to differentiate into cardiomyocytes(J. Clinical Investigation, 103: 10-18 (1999)). This report suggeststhat the cell therapy which comprises transplanting cells which areobtained from bone marrow fluid taken from a patient followed by invitro expansion and drug treatment to the damaged part of the patient'sheart can be a practical medical treatment (J. Clinical Investigation,103: 591-592 (1999)). However, this report merely indicates that a partof the immortalized cells established from the bone marrow of an adultmouse can differentiate into cardiomyocytes. Furthermore, the reportfails to isolate, selectively proliferate, and efficiently differentiatethe adult bone marrow cells having the potential to differentiate intocardiomyocytes (J. Clinical Investigation, 103: 591-592 (1999)).

[0010] Antibodies which recognize various surface antigens are used toisolate the target cells from the tissue of vital body. For example, itis known that immature hematopoietic stem cells have the characteristicsof CD34+/CD38-/HLA-DR-/CD90 (Thy-1)+, and CD38 is expressed whileCD90(Thy-1) disappears in the process of differentiation (Protein,Nucleic Acid, Enzyme, 45: 13, 2056-2062 (2000)). In vascular endothelialcells, markers such as CD34, CD31, Flk-1, Tie-2, E-selectin, etc. areexpressed (Molecular Cardiovascular Disease, 1(3): 294-302 (2000)). Inbone marrow mesenchymal stem cells, markers such as CD90, CD105, CD140,etc. are expressed (Science, 284: 143-147 (1999); Science, 284:1168-1170 (1999)). However, no surface marker of stem cells capable ofinducing both myocardium and vascular endothelial cells is known.

DISCLOSURE OF THE INVENTION

[0011] Under the circumstances, a need exists for the development oftherapy for heart diseases which therapy is safer and more establishedthan those currently available. It is useful to select cells having thepotential to differentiate into cardiomyocytes from a vital tissue suchas bone marrow cells or the like or umbilical blood and to control thegrowth or differentiation of the cells for the development ofmyocardium-regenerating therapy using vital cells such as bonemarrow-derived cells or the like or umbilical blood. For this purpose,it is necessary to separate the cells having the potential todifferentiate into cardiomyocytes and to identify cytokines ortranscription factors participating in the growth or differentiation ofsuch cells.

[0012] The present inventors have made intensive studies aiming atsolving the above problems and have obtained the following results.Specifically, various cell lines were obtained by separating mouse bonemarrow-derived cells to single cell level. Then, various cell lines havecharacterized by their potential to differentiate into cardiomyocytes bytreating each cell line with 5-azacytidine. Next, by labeling the thusobtained cell line using a retrovirus vector which expresses a GFP(green fluorescent protein) and tracing the cells using a fluorescencemicroscope, it has been found that the bone marrow-derived cells arepluripotent stem cells which can differentiate into at least twodifferent cells, i.e., cardiomyocytes and adipocytes. Furthermore, ithas been found that the stem cells can be differentiated intocardiomyocytes, adipocytes and skeletal muscle cells stochastically byaddition of not only 5-azacytidine but also other genomicDNA-demethylating agents, such as DMSO (dimethyl sulfoxide), indicatingthat demethylation of genomic DNA is effective in inducing thedifferentiation of bone marrow-derived cells into cardiomyocytes.Moreover, it was found that the expression of myocardium-specific genes,ANP (atrral natriuretic peptide) and cTnI (cardiac Troponin I), can beexpressed in the bone marrow-derived cells by adding at least onecytokine of four cytokines, FGF-8, ET1, midkine and BMP4, combined with5-azacytidine. Also, it was found that differentiation of the bonemarrow-derived cells into cardiomyocytes can be promoted about 50-foldby the forced expression of two transcriptional factors, Nkx2.5 andGATA4, in these bone marrow-derived cells using virus vectors followedby 5-azacytidine treatment. Furthermore, it was found that theexpression of ANP and cTnI, which are myocardium-specific genes, in thebone marrow-derived cells can be specifically promoted by culturingthese bone marrow-derived cells in a culture dish coated with acardiomyocyte-derived extracellular substrate. Moreover, it was foundthat the formation of myocardium from the bone marrow-derived cells canbe about 10 times or more promoted by co-culturing the bonemarrow-derived cells together with primarily cultured cells derived frommyocardium. Moreover, it was found that differentiation of the bonemarrow-derived cells into cardiomyocytes can be promoted about 500-foldwhen the forced expression of two transcription factors Nkx2.5 and GATA4in the bone marrow-derived cells using virus vectors and co-culturingthese cells with cardiomyocytes were combined.

[0013] Subsequently, the differentiation potency of the bonemarrow-derived cells was examined by a transplantation experiment.First, the bone marrow-derived cells were transplanted into an adultmouse heart and it was thus found that these bone marrow-derived cellswere differentiated into myocardia and vessels. Furthermore, the bonemarrow-derived cells were transplanted into an adult mouse muscle and itwas thus found that these bone marrow-derived cells could form skeletalmuscles. When the bone marrow-derived cells were transplanted into amouse blastocyst, tissues derived from these transplanted cells wereformed in the central nervous system, liver and heart of the mouse. Thecentral nervous system, liver and heart are tissues of the ectoderm,endoderm and mesoderm, respectively.

[0014] These results indicate that the bone marrow-derived cells foundin the present invention have properties different from those possessedby hematopoietic stem cells which are differentiated into onlyhematopoietic stem tissue present in bone marrow and from thosepossessed by mesenchymal stem cell which are differentiated into onlydorsal mesoderm tissue such as skeletal muscle, adipocytes, bone and thelike known in the art, that is, a totipotency of differentiating intoall of the three germ layers including the ectoderm, mesoderm andendoderm.

[0015] Furthermore, the inventors analyzed the expression of surfaceantigens of bone marrow-derived cells using antibodies which recognizehematopoietic cell surface antigens, CD34, CD117, CD14, CD45, CD90,Sca-1, Ly6c and Ly6g, antibodies which recognize vascular endothelialcell surface antigens, Flk-1, CD31, CD105 and CD144, antibodies whichrecognize a mesenchymal cell surface antigen, CD140, antibodies whichrecognize integrin surface antigens, CD49b, CD49d, CD29 and CD41, andantibodies which recognize matrix receptors, CD54, CD102, CD106 andCD44, and the like in these bone marrow cells of the present inventionand thus found that they are totipotential stem cells exhibiting a quitenovel expression form having been unknown, thereby completing thepresent invention.

[0016] Specifically, the present invention provides the following(1)-(91):

[0017] (1) A cell which has been isolated from a living tissue orumbilical blood, and which has the potential to differentiate into atleast a cardiomyocyte.

[0018] (2) The cell according to (1), wherein the living tissue is bonemarrow.

[0019] (3) The cell according to (1) or (2), wherein the cell is amultipotential stem cell.

[0020] (4) The cell according to any one of (1) to (3), wherein the cellis a multipotential stem cell which differentiates into at least acardiomyocyte and a vascular endothelial cell.

[0021] (5) The cell according to any one of (1) to (4), wherein the cellis a multipotential stem cell which differentiates into at least acardiomyocyte, an adipocyte, a skeletal muscle cell, an osteoblast, anda vascular endothelial cell.

[0022] (6) The cell according to any one of (1) to (5), wherein the cellis a multipotential stem cell which differentiates into at least acardiomyocyte, an adipocyte, a skeletal muscle cell, an osteoblast, avascular endothelial cell, a nervous cell, and a hepatic cell.

[0023] (7) The cell according to any one of (1) to (3), wherein the cellis a multipotential stem cell which differentiates into any cell inadult tissues.

[0024] (8) The cell according to any one of (1) to (7), wherein the cellis CD117-positive and CD140-positive.

[0025] (9) The cell according to (8), wherein the cell is furtherCD34-positive.

[0026] (10) The cell according to (9), wherein the cell is furtherCD144-positive.

[0027] (11) The cell according to (9), wherein the cell is furtherCD140-negative.

[0028] (12) The cell according to (8), wherein the cell isCD34-negative.

[0029] (13) The cell according to (12), wherein the cell is furtherCD144-positive.

[0030] (14) The cell according to (12), wherein the cell is furtherCD144-negative.

[0031] (15) The cell according to (10), wherein the cell is furtherCD14-negative, CD45-negative, CD90-negative, Flk-1-negative,CD31-negative, CD105-negative, CD49b-negative, CD49d-negative,CD29-positive, CD54-negative, CD102-negative, CD106-negative, andCD44-positive.

[0032] (16) The cell according to (11), wherein the cell is furtherCD14-negative, CD45-negative, CD90-negative, Flk-1-negative,CD31-negative, CD105-negative, CD49b-negative, CD49d-negative,CD29-positive, CD54-negative, CD102-negative, CD106-negative, andCD44-positive.

[0033] (17) The cell according to (12), wherein the cell is furtherCD14-negative, CD45-negative, CD90-negative, Flk-1-negative,CD31-negative, CD105-negative, CD49b-negative, CD49d-negative,CD29-positive, CD54-negative, CD102-negative, CD106-negative, andCD44-positive.

[0034] (18) The cell according to (13), wherein the cell is furtherCD14-negative, CD45-negative, CD90-negative, Flk-1-negative,CD31-negative, CD105-negative, CD49b-negative, CD49d-negative,CD29-positive, CD54-negative, CD102-negative, CD106-negative, andCD44-positive.

[0035] (19) The cell according to (1), which does not take up Hoechst33342.

[0036] (20) A cardiomyocyte precursor which differentiates into onlycardiomyocyte induced from the cell according to any one of (1) to (19).

[0037] (21) The cell according to any one of (1) to (20), which has thepotential to differentiate into a ventricular cardiac muscle cell.

[0038] (22) The cell according to any one of (1) to (20), which has thepotential to differentiate into a sinus node cell.

[0039] (23) The cell according to any one of (1) to (20), wherein thevital tissue or umbilical blood is derived from a mammal.

[0040] (24) The cell according to (23), wherein the mammal is selectedfrom the group consisting of a mouse, a rat, a guinea pig, a hamster, arabbit, a cat, a dog, a sheep, a swine, cattle, a goat and a human.

[0041] (25) The cell according to any one of (1) to (8), which is mousebone marrow-derived multipotential stem cell BMSC (FERM BP-7043).

[0042] (26) The cell according to any one of (1) to (25), which has thepotential to differentiate into a cardiomyocyte by demethylation of achromosomal DNA of the cell.

[0043] (27) The cell according to (26), wherein the demethylation iscarried out by at least one selected from the group consisting ofdemethylase, 5-azacytidine, and dimethyl sulfoxide, DMSO.

[0044] (28) The cell according to (27), wherein the demethylasecomprises the amino acid sequence represented by SEQ ID NO:1.

[0045] (29) The cell according to any one of (1) to (28), wherein thedifferentiation is accelerated by a factor which is expressed in acardiogenesis region of a fetus or a factor which acts ondifferentiation into a cardiomyocyte in a cardiogenesis stage of afetus.

[0046] (30) The cell according to (29), wherein the factor which isexpressed in a cardiogenesis region of a fetus or the factor which actson differentiation into a cardiomyocyte in a cardiogenesis stage of afetus is at least one selected from the group consisting of a cytokine,an adhesion molecule, a vitamin, a transcription factor, and anextracellular matrix.

[0047] (31) The cell according to (30), wherein the cytokine is at leastone selected from the group consisting of a platelet-derived growthfactor, PDGF; a fibroblast growth factor-8, FGF-8; an endothelin 1, ET1;a midkine; and a bone morphogenetic factor, BMP-4.

[0048] (32) The cell according to (31), wherein the PDGF, FGF-8, ET1,midkine, and BMP-4 comprise the amino acid sequence represented by SEQID NO:3 or 5, the amino acid sequence represented by SEQ ID NO:64, theamino acid sequence represented by SEQ ID NO:66, the amino acid sequencerepresented by SEQ ID NO:68, and the amino acid sequence represented bySEQ ID NO:70, respectively.

[0049] (33) The cell according to (30), wherein the adhesion molecule isat least one selected from the group consisting of a gelatin, a laminin,a collagen, and a fibronectin.

[0050] (34) The cell according to (30), wherein the vitamin is retinoicacid.

[0051] (35) The cell according to (30), wherein the transcription factoris at least one selected from the group consisting of Nkx2)5/Csx, GATA4,MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5, andMesP1.

[0052] (36) The cell according to (35), wherein the Nkx2)5/Csx, GATA4,MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5, andMesP1 comprise the amino acid sequence represented by SEQ ID NO:9, theamino acid sequence represented by SEQ ID NO:11, the amino acid sequencerepresented by SEQ ID NO:13, the amino acid sequence represented by SEQID NO:15, the amino acid sequence represented by SEQ ID NO:17, the aminoacid sequence represented by SEQ ID NO:19, the amino acid sequencerepresented by SEQ ID NO:21, the amino acid sequence represented by SEQID NO:23, the amino acid sequence represented by SEQ ID NO:25, the aminoacid sequence represented by SEQ ID NO:27, the amino acid sequencerepresented by SEQ ID NO:29, and the amino acid sequence represented bySEQ ID NO:62, respectively.

[0053] (37) The cell according to (30), wherein the extracellular matrixis an extracellular matrix derived from a cardiomyocyte.

[0054] (38) The cell according to any one of (1) to (28), wherein thedifferentiation is inhibited by a fibroblast growth factor-2, FGF-2.

[0055] (39) The cell according to (38), wherein the FGF-2 comprises theamino acid sequence represented by SEQ ID NO:7 or 8.

[0056] (40) The cell according to any one of (1) to (28), which iscapable of differentiating into a cardiomyocyte or a blood vessel bytransplantation into a heart.

[0057] (41) The cell according to any one of (1) to (28), which iscapable of differentiating into a cardiac muscle by transplantation intoa blastocyst or by co-culturing with a cardiomyocyte.

[0058] (42) The cell according to any one of (1) to (28), which iscapable of differentiating into an adipocyte by an activator of anuclear receptor, PPAR-Y.

[0059] (43) The cell according to (42), wherein the activator is acompound having a thiazolidione skeleton.

[0060] (44) The cell according to (43), wherein the compound is at leastone selected from the group consisting of troglitazone, pioglitazone,and rosiglitazone.

[0061] (45) The cell according to any one of (1) to (28), which iscapable of differentiating into a nervous cell by transplantation into ablastocyst or by transplantation into an encephalon or a spinal cord.

[0062] (46) The cell according to any one of (1) to (28), which iscapable of differentiating into a hepatic cell by transplantation into ablastocyst or by transplantation into a liver.

[0063] (47) A method for differentiting the cell according to any one of(1) to (28) into a cardiac muscle, comprising using a chromosomalDNA-dimethylating agent.

[0064] (48) A method for redifferentiating the cell according to (9)into the cell according to (12), comprising using a chromosomalDNA-dimethylating agent.

[0065] (49) A method for redifferentiating a cell which isCD117-negative and CD140-positive into the cell according to (8),comprising using a chromosomal DNA-dimethylating agent.

[0066] (50) The method according to (48) or (49), wherein thechromosomal DNA-dimethylating agent is selected from the groupconsisting of a demethylase, 5-azacytidine, and DMSO.

[0067] (51) The method according to (50), wherein the demethylasecomprises the amino acid sequence represented by SEQ ID NO:1.

[0068] (52) A method for differentiating the cell according to any oneof (1) to (28) into a cardiac muscle, comprising using a factor which isexpressed in a cardiogenesis region of a fetus or a factor which acts ondifferentiation into a cardiomyocyte in a cardiogenesis stage of afetus.

[0069] (53) The method according to (52), wherein the factor which isexpressed in a cardiogenesis region of a fetus or the factor which actson differentiation into a cardiomyocyte in a cardiogenesis stage of afetus is at least one selected from the group consisting of a cytokine,an adhesion molecule, a vitamin, a transcription factor, and anextracellular matrix.

[0070] (54) The method according to (53), wherein the cytokine is atleast one selected from the group consisting of a platelet-derivedgrowth factor, PDGF; a fibroblast growth factor-8, FGF-8; an endothelin1, ET1; a midkine; and a bone morphogenetic factor, BMP-4.

[0071] (55) The method according to (54), wherein the PDGF, FGF-8, ET1,midkine, and BMP-4 comprise the amino acid sequence represented by SEQID NO:3 or 5, the amino acid sequence represented by SEQ ID NO:64, theamino acid sequence represented by SEQ ID NO:66, the amino acid sequencerepresented by SEQ ID NO:68, and the amino acid sequence represented bySEQ ID NO:70, respectively.

[0072] (56) The method according to (53), wherein the adhesion moleculeis at least one selected from the group consisting of a gelatin, alaminin, a collagen, and a fibronectin.

[0073] (57) The method according to (53), wherein the vitamin isretinoic acid.

[0074] (58) The method according to (53), wherein the transcriptionfactor is at least one selected from the group consisting of Nkx2)5/Csx,GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3,TEF-5, and MesP1.

[0075] (59) The method according to (58), wherein the Nkx2)5/Csx, GATA4,MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5, andMesP1 comprise the amino acid sequence represented by SEQ ID NO:9, theamino acid sequence represented by SEQ ID NO:11, the amino acid sequencerepresented by SEQ ID NO:13, the amino acid sequence represented by SEQID NO:15, the amino acid sequence represented by SEQ ID NO:17, the aminoacid sequence represented by SEQ ID NO:19, the amino acid sequencerepresented by SEQ ID NO:21, the amino acid sequence represented by SEQID NO:23, the amino acid sequence represented by SEQ ID NO:25, the aminoacid sequence represented by SEQ ID NO:27, the amino acid sequencerepresented by SEQ ID NO:29, the amino acid sequence represented by SEQID NO:62, respectively.

[0076] (60) The method according to (53), wherein the extracellularmatrix is an extracellular matrix derived from a cardiomyocyte. (61) Amethod for differentiating the cell according to any one of (1) to (28)into an adipocyte, comprising using an activator of a nuclear receptor ,PPAR-γ.

[0077] (62) The method according to (61), wherein the activator is acompound having a thiazolidione skeleton.

[0078] (63) The method according to (62), wherein the compound is atleast one selected from the group consisting of troglitazone,pioglitazone, and rosiglitazone.

[0079] (64) A myocardium-forming agent, comprising, as an activeingredient, a chromosomal DNA-demethylating agent.

[0080] (65) The myocardium-forming agent according to (64), wherein thechromosomal DNA-demethylating agent is at least one selected from thegroup consisting of a demethylase, 5-azacytidine, and DMSO.

[0081] (66) The myocardium-forming agent according to (65), wherein thedemethylase comprises the amino acid sequence represented by SEQ IDNO:1.

[0082] (67) A myocardium-forming agent, comprising, as an activeingredient, a factor which is expressed in a cardiogenesis region of afetus or a factor which acts on differentiation into a cardiomyocyte ina cardiogenesis stage of a fetus.

[0083] (68) The myocardium-forming agent according to (67), wherein thefactor which is expressed in a cardiogenesis region of a fetus or thefactor which acts on differentiation into a cardiomyocyte in acardiogenesis stage of a fetus is at least one selected from the groupconsisting of a cytokine, an adhesion molecule, a vitamin, atranscription factor, and an extracellular matrix.

[0084] (69) The myocardium-forming agent according to (68), wherein thecytokine is at least one selected from the group consisting of aplatelet-derived growth factor, PDGF; a fibroblast growth factor-8,FGF-8; an endothelin 1, ET1; a midkine; and a bone morphogenetic factor,BMP-4.

[0085] (70) The myocardium-forming agent according to (69), wherein thePDGF, FGF-8, ET1, midkine, and BMP-4 comprise the amino acid sequencerepresented by SEQ ID NO:3 or 5, the amino acid sequence represented bySEQ ID NO:64, the amino acid sequence represented by SEQ ID NO:66, theamino acid sequence represented by SEQ ID NO:68, and the amino acidsequence represented by SEQ ID NO:70, respectively.

[0086] (71) The myocardium-forming agent according to (68), wherein theadhesion molecule is selected from the group consisting of a gelatin, alaminin, a collagen, and a fibronectin.

[0087] (72) The myocardium-forming agent according to (71), wherein thevitamin is retinoic acid.

[0088] (73) The myocardium-forming agent according to (68), wherein thetranscription factor is at least one selected from the group consistingof Nkx2)5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND,TEF-1, TEF-3, TEF-5, and MesP1.

[0089] (74) The myocardium-forming agent according to (73), wherein theNkx2)5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1,TEF-3, TEF-5, and MesP1 comprise the amino acid sequence represented bySEQ ID NO:9, the amino acid sequence represented by SEQ ID NO:11, theamino acid sequence represented by SEQ ID NO: 13, the amino acidsequence represented by SEQ ID NO:15, the amino acid sequencerepresented by SEQ ID NO:17, the amino acid sequence represented by SEQID NO:19, the amino acid sequence represented by SEQ ID NO:21, the aminoacid sequence represented by SEQ ID NO:23, the amino acid sequencerepresented by SEQ ID NO:25, the amino acid sequence represented by SEQID NO:27, the amino acid sequence represented by SEQ ID NO:29, and theamino acid sequence represented by SEQ ID NO:62, respectively.

[0090] (75) The myocardium-forming agent according to (68), wherein theextracellular matrix is an extracellular matrix derived from acardiomyocyte.

[0091] (76) A method for regenerating a heart damaged by a heartdisease, comprising using the cell according to any one of (1) to (46).

[0092] (77) An agent for cardiac regeneration, comprising, as an activeingredient, the cell according to any one of (1) to (46).

[0093] (78) A method for specifically transfecting a wild-type genecorresponding to a mutant gene in a congenital genetic disease to amyocardium, comprising using the cell according to any one of (1) to(46) into which the wild-type gene corresponding to a mutant gene in acongenital genetic disease of a heart has been introduced.

[0094] (79) A therapeutic agent for a heart disease, comprising, as anactive ingredient, the cell according to any one of (1) to (46) intowhich a wild-type gene corresponding to a mutant gene in a congenitalgenetic disease of a heart has been introduced.

[0095] (80) A method for producing an antibody which specificallyrecognizes the cell according to any one of (1) to (46), comprisingusing the cell as an antigen.

[0096] (81) A method for isolating a cell having the potential todifferentiate into a cardiomyocyte according to any one of (1) to (46),comprising using an antibody obtained by the method according to (80).

[0097] (82) A method for obtaining a surface antigen specific for thecell according to any one of (1) to (46), comprising using the cell.

[0098] (83) A method for screening a factor which proliferates the cellaccording to any one of (1) to (46), comprising using the cell.

[0099] (84) A method for screening a factor which induces the cellaccording to any one of (1) to (46) to differentiate into acardiomyocyte, comprising using the cell.

[0100] (85) A method for screening a factor which immortalizes the cellaccording to any one of (1) to (46), comprising using the cell.

[0101] (86) A method for immortalizing the cell according to any one of(1) to (46), comprising expressing a telomerase in the cell.

[0102] (87) The method according to (86), wherein the telomerasecomprises the amino acid sequence represented by SEQ ID NO:31.

[0103] (88) A therapeutic agent for a heart disease, comprising, as anactive ingredient, the cell according to any one of (1) to (46) whichhas been immortalized by expressing a telomerase.

[0104] (89) The therapeutic agent according to (88), wherein thetelomerase comprises the amino acid sequence represented by SEQ IDNO:31.

[0105] (90) A culture supernatant comprising the cell according to anyone of (1) to (46).

[0106] (91) A method for inducing the cell according to any one of (1)to (46) to differentiate into a cardiomyocyte, comprising using theculture supernatant according to (90).

[0107] The cells having the potential to differentiate intocardiomyocytes according to the present invention can be isolated fromadult tissues such as bone marrow, muscle, brain, pancreas, liver andkidney or umbilical blood, and preferred examples include bone marrowand umbilical blood.

[0108] Any cell can be used as the pluripotent stem of the presentinvention, so long as it has the potential to differentiate intocardiomyocytes and other cells. Preferable examples thereof includecells having the potential to differentiate into at leastcardiomyocytes, adipocytes, skeletal muscle cells and osteoblasts; cellshaving the potential to differentiate into at least cardiomyocyte andvascular endothelial cells; cells having the potential to differentiateinto at least cardiomyocytes, adipocytes, skeletal muscle cells,osteoblasts and vascular endothelial cells; and cells having thepotential to differentiate into at least cardiomyocytes, adipocytes,skeletal muscle cells, vascular endothelial cells, osteoblasts, neuralcells and hepatocytes.

[0109] Also, even if cells originally have the potential todifferentiate into adipocytes, skeletal muscle cells and osteoblasts butdo not have the potential to differentiate into cardiomyocytes, thosecells to which the potential to differentiate into cardiomyocytes can beadded by the following induction method or the like, are included in theinvention.

[0110] The cells of the present invention having the potential todifferentiate into cardiomyocytes include cells which are CD117-positiveand CD140-positive. The cells which are CD117-positive andCD140-positive preferably cells which are CD34-positive, CD117-positiveand CD140-positive, and cells which are CD34-negative, CD117-positiveand CD140-positive; more preferably cells which are CD144-positive,CD34-positive, CD117-positive and CD140-positive, cell which areCD144-negative, CD34-positive, CD117-positive and CD140-positive, cellswhich are CD144-positive, CD34-negative, CD117-positive andCD140-positive, and cells which are CD144-negative, CD34-negative,CD117-positive and CD140-positive; still more preferably cells which areCD34-positive, CD117-positive, CD14-negative, CD45-negative,CD90-negative, Flk-1-negative, CD31-negative, CD105-negative,CD144-positive, CD140-positive, CD49b-negative, CD49d-negative,CD29-positive, CD54-negative, CD102-negative, CD106-negative andCD44-positive, cells which are CD34-positive, CD117-positive,CD14-negative, CD45-negative, CD90-negative, Flk-1-negative,CD31-negative, CD105-negative, CD144-negative, CD140-positive,CD49b-negative, CD49d-negative, CD29-positive, CD54-negative,CD102-negative, CD106-negative and CD44-positive, cells which areCD34-negative, CD117-positive, CD14-negative, CD45-negative,CD90-negative, Flk-1-negative, CD31-negative, CD105-negative,CD144-positive, CD140-positive, CD49b-negative, CD49d-negative,CD29-positive, CD54-negative, CD102-negative, CD106-negative andCD44-positive, and cells which are CD34-positive, CD117-positive,CD14-negative, CD45-negative, CD90-negative, Flk-1-negative,CD31-negative, CD105-negative, CD144-negative, CD140-positive,CD49b-negative, CD49d-negative, CD29-positive, CD54-negative,CD102-negative, CD106-negative and CD44-positive. The cells which areCD117-positive and CD140-positive include mouse marrow multipotentialstem cells, BMSC. Mouse bone marrow-derived pluripotent stem cells(BMSC) have been deposited on Feb. 22, 2000, in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology (Higashi 1-1-3, Tsukuba-shi, Ibaraki, Japan) as FERM BP-7043.

[0111] Examples of the cells which originally have the potential todifferentiate into adipocytes, skeletal muscle cells and osteoblasts butdo not have the potential to differentiate into cardiomyocytes, to whichthe potential to differentiate into heart muscle cells can be added bythe following induction method or the like include cells which areCD117-negative and CD140-positive, preferably cells which areCD144-negative, CD34-negative, CD117-negative and CD140-positive, morepreferably cells which are CD34-negative, CD117-negative, CD14-positive,CD45-negative, CD90-negative, Flk-1-negative, CD31-negative,CD105-negative, CD144-negative, CD140-positive, CD49b-positive,CD49d-negative, CD29-positive, CD54-positive, CD102-negative,CD106-positive and CD44-positive. KUM2 cells can be exemplified as thecells which are CD34-negative, CD117-negative, CD14-positive,CD45-negative, CD90-negative, Flk-1-negative, CD31-negative,CD105-negative, CD144-negative, CD140-positive, CD49b-positive,CD49d-negative, CD29-positive, CD54-positive, CD102-negative,CD106-positive and CD44-positive.

[0112] As the species of the vital tissue or umbilical blood used in theinvention, vertebrate animals, preferably warm blooded animals, and morepreferably mammals such as mouse, rat, guinea pig, hamster, rabbit, cat,dog, sheep, pig, cattle, goat, monkey and human are used. Those derivedfrom a human is preferred for human therapeutic use.

[0113] Any adult tissue or umbilical blood can be used, so long as it isderived from the above animal. In therapeutic use for the human body, itis preferred to employ those derived from humans.

[0114] Myocardial cells can be obtained by isolating cells having thepotential to differentiate into cardiomyocytes from an adult tissue orumbilical blood of a mammal, such as mouse, rat or human, culturingthese cells and then inducing the differentiation of cells having thepotential to differentiate into cardiomyocytes.

[0115] The differentiation into not only cardiomyocytes but alsovascular endothelial cells, smooth muscles, skeletal muscle cells,adipocytes, bones, cartilages, pancreatic endocrine cells, pancreaticexocrine cells, hepatocytes, glomerular cells, renal tubular cells,neurons, glial cells, oligodendrocytes, etc. can be induced using thepluripotent stem cell to obtain various cells.

[0116] Now, the present invention will be described in greater detail.

[0117] 1. Isolation of Cells Having the Potential to Differentiate intoCardiomyocytes

[0118] The cells having the potential to differentiate intocardiomyocytes according to the present invention can be isolated fromany tissue (for example, an adult tissue, umbilical blood), so long ascells having the potential to differentiate into cardiomyocytes can beobtained. Next, a method for isolating cells having the potential todifferentiate into cardiomyocytes from bone marrow will be illustrated.

[0119] (1) Method for Isolating Bone Marrow Cells Having the Potentialto Differentiate into Cardiomyocytes

[0120] The method for obtaining human cells having the potential todifferentiate into cardiomyocytes from bone marrow is not particularlylimited, so long as it is a safe and efficient method. For example, themethod described in S. E. Haynesworth, et al., Bone, 13: 81 (1992) canbe employed.

[0121] Bone marrow puncture is conducted by sternal or iliac puncture.After skin disinfection of the part for puncture, a donor is subjectedto local anesthesia. Particularly, subpeiosteum is thoroughlyanesthetized. The inner tube of a bone marrow puncture needle is pulledout and a 10 ml syringe containing 5000 units of heparin is attached tothe needle. A required amount, normally 10-20 ml, of the bone marrowfluid is quickly taken by suction and the puncture needle is removed,followed by pressure hemostasis for about 10 minutes. The obtained bonemarrow fluid is centrifuged at 1000 × g to recover bone marrow cells,which are then washed with PBS (phosphate buffered saline). After thiscentrifugation step is repeated twice, the obtained bone marrow cellsare suspended in a cell culture medium such as A-MEM (a-modification ofMEM), DMEM (Dulbecco's modified MEM) or IMDM (Isocove's modifiedDulbeccos's medium) each containing 10% FBS (fetal bovine serum) toprepare a bone marrow cell suspension.

[0122] For the isolation of the bone marrow cells having the potentialto differentiate into cardiomyocytes from the obtained bone marrow cellsuspension, any method can be employed, so long as it is effective forremoving other cells existing in the cell suspension such ashematocytes, hematopoietic stem cells, vascular stem cells andfibroblasts. For example, based on the method described in M. F.Pittenger et al., Science, 284: 143 (1999), the desired cells can beisolated by subjecting the cell suspension layered over Percoll havingthe density of 1.073 g/ml to centrifugation at 1100 × g for 30 minutes,and the cells on the interface are recovered. Furthermore, a bone marrowcell mixture containing the cells having the potential to differentiateinto cardiomyocytes can be obtained by mixing the above cell suspensionwith an equal amount of Percoll solution diluted to {fraction (9/10)}with 10× PBS, followed by centrifugation at 20000 × g for 30 minutes,and recovering the fraction having the density of 1.075-1.060.

[0123] The thus obtained bone marrow cell mixture containing the bonemarrow cells having the potential to differentiate into cardiomyocytesis diluted into single cell using 96-well culture plates to prepare anumber of clones respectively derived from single cells. The cloneshaving the potential to differentiate into cardiomyocyte can be selectedby the observation of spontaneously beating cells generated by thetreatment to induce cardiomyocytes from the cells having the potentialto differentiate into cardiomyocytes described below.

[0124] Rat- or mouse-derived bone marrow cells having the potential todifferentiate into cardiomyocytes can be obtained, for example, in thefollowing manner. A rat or a mouse is sacrificed by cervical dislocationand thoroughly disinfected with 70% ethanol. After the skin on the femurand quadriceps femuris are excised, the femur is put out of the kneejoint with scissors and the muscle on the back side of the femur isremoved. Then, the femur is put out of the hip joint with scissors andtaken out. After the muscle on the femur is removed with scissors ascompletely as possible, the femur is cut at both ends using scissors. Aneedle having a size appropriate for the thickness of the bone isattached to a 2.5 ml syringe containing about 1.5 ml of a cell culturemedium such as α-MEM, DMEM or IMDM each containing 10% FBS followed byinjecting into the pore of femur. The needle of the syringe is put intothe femur from the cut end of the knee joint side and the culture mediumis injected into bone marrow, whereby bone marrow cells are pressed outof the bone from the cut end of the hip joint side. The thus obtainedbone marrow cells are suspended in a culture medium by pipetting. Thebone marrow cells having the potential to differentiate intocardiomyocytes can be isolated from the resulting cell suspension in thesame manner as in the above isolation of the human bone marrow cells.

[0125] (2) Method for Isolating Cells having the Potential toDifferentiate into Cardiomyocytes from Tissue other than Bone Marrow

[0126] According to the separation method using antibodies as describedin 12 hereinafter, cells having the potential to differentiate intocardiomyocytes can be obtained form tissues other than bone marrow.

[0127] Preferred examples of the tissues other than bone marrow includeumbilical blood. More specifically, it can be isolated in the followingmethod.

[0128] First, umbilical blood is separated from the cord, followed byaddition of heparin to give a final concentration of 500 units/ml. Afterthoroughly mixing, cells are separated from the umbilical blood bycentrifugation and re-suspended in a cell culture medium, such as α-MEM(a-modified MEM), DMEM (Dulbecco's modified MEM) or IMDM (Isocove'smodified Dulbecco's medium), each containing 10% FBS. From the cellsuspension thus obtained, cells having the potential to differentiateinto cardiomyocytes can be separated using the antibodies describedbelow.

[0129] 2. Methods for Culturing the Cells having the Potential toDifferentiate into Cardiomyocytes

[0130] The cells having the potential to differentiate intocardiomyocytes isolated by the methods described in the above 1 can beusually cultured using media of known compositions (Technical Standardof Tissue Culture, Third Edition, Asakura Shoten (1996)). Preferredmedia are cell culture media such as α-MEM, DMEM and IMDM supplementedwith a serum such as 5-20% bovine serum. Culturing can be carried outunder any conditions suitable for cell culture, but is preferablycarried out at a temperature of 33-37° C. in an incubator filled with5-10% carbon dioxide gas. It is preferred to culture the cells havingthe potential to differentiate into cardiomyocytes in a plastic culturedish used for ordinary tissue culture so that the grown cells adhere tothe dish. When cells become confluent on the dish, the medium is removedand a trypsin-EDTA solution is added to suspend the cells therein. Thesuspended cells may be washed with PBS or a medium for culturing thecells, diluted 5-20 times with the medium and then added to anotherculture dish for subculture.

[0131] 3. Methods for Inducing Cardiomyocytes from Cells having thePotential to Differentiate into Cardiomyocytes

[0132] The methods for inducing cardiomyocytes from the cells having thepotential to differentiate into cardiomyocytes include the following:(1) induction of differentiation by the treatment with aDNA-demethylating agent, (2) induction of differentiation using a factorwhich is expressed in the cardiogenesis region of a fetus or a factorwhich controls differentiation into cardiomyocytes in the cardiogenesisstage of a fetus, and (3) induction of differentiation using a culturesupernatant of the cells having the potential to differentiate intocardiomyocytes or cardiomyocytes differentiated from the cells.Cardiomyocytes can be induced from the cells having the potential todifferentiate into cardiomyocytes using such a method alone or incombination. Also, according to these methods, even mesenchymal cellswhich originally do not have the potential to differentiate intocardiomyocytes can be differentiated into cells having the potential todifferentiate into cardiomyocytes, and cardiomyocytes can be induced.

[0133] Any DNA-demethylating agent can be used, so long as it is acompound which causes demethylation of DNA. Suitable DNA-demethylatingagents include demethylase which is an enzyme which specifically removesthe methylation of the cytosine residue in the GpC sequence in achromosomal DNA, 5-azacytidine (hereinafter referred to as “5-aza-C”)and DMSO (dimethyl sulfoxide). Examples of the demethylase enzymesinclude demethylase having the amino acid sequence represented by SEQ IDNO:1 (Nature, 397: 579-583 (1999)). Differentiation can be induced bythe treatment with a DNA-demethylating agent, for example, in thefollowing manner.

[0134] The cells having the potential to differentiate intocardiomyocytes are cultured in the presence of 3 μmol/l to 10 μmol/l of5-aza-C for 24 hours. After 5-aza-C is removed by replacing the culturesupernatant with a fresh medium, the cells are cultured for further 2-3weeks to obtain cardiomyocytes. The cardiomyocytes produced by culturingfor 2-3 weeks are mainly sinus node cells, but culturing for more than 4weeks induces differentiation into ventricular cardiomyocytes.

[0135] Examples of the factors which are expressed in the cardiogenesisregion of a fetus and the factors which act on differentiation intocardiomyocytes in the cardiogenesis stage of a fetus include cytokines,vitamins, adhesion molecules and transcription factors.

[0136] Any cytokine can be used, so long as it stimulates thecardiomyogenic differentiation of the cells having the potential todifferentiate into cardiomyocytes in the cardiogenesis stage.

[0137] The examples include platelet-derived growth factor (hereinafterreferred to as “PDGF”), fibroblast growth factor 8 (FGF8), endothelin 1(ET1), midkine, and bone morphogenic protein 4 (BMP4). Preferredexamples of the PDGF include PDGF A, PDGF B, PDGF C and the like, andspecific examples include those the amino acid sequences represented bySEQ ID NOS:3 and 5. Preferred examples of the FGF8, ET1, midkine, BMP4include the amino acid sequence represented by SEQ ID NO:64, the aminoacid sequence represented by SEQ ID NO:66, the amino acid sequencerepresented by SEQ ID NO:68, and the amino acid sequence represented bySEQ ID NO:70, respectively. The cytokine can be used, e.g., at aconcentration of 10 to 40 ng/ml.

[0138] It is also possible to stimulate the cardiomyogenicdifferentiation of the cells having the potential to differentiate intocardiomyocytes into cardiomyocytes in the cardiogenesis stage using aninhibitor against a cytokine which suppresses the cardiomyogenicdifferentiation.

[0139] The cytokines which suppress the cardiomyogenic differentiationinclude fibroblast growth factor-2 (hereinafter referred to as“IFGF-2”), specifically, FGF-2 having the amino acid sequencerepresented by SEQ ID NO:7 or 8.

[0140] The inhibitors against the cytokines which suppress thecardiomyogenic differentiation include substances which inhibit thesignal transduction of the cytokines, such as antibodies and lowmolecular weight compounds which neutralize the cytokines activities.

[0141] Any vitamin can be used, so long as it stimulates thecardiomyogenic differentiation of the cells having the potential todifferentiate into cardiomyocytes in the cardiogenesis stage. Retinoicacid can be used, e.g., at a concentration of 10⁻⁹ M.

[0142] Any adhesion molecule can be used, so long as it is expressed inthe cardiogenesis region in the cardiogenesis stage. Examples includeextracellular matrices such as gelatin, laminin, collagen, fibronectinand the like. For example, the cardiomyogenic differentiation of thecells having the potential to differentiate into cardiomyocytes can bestimulated by culturing the cells on a culture dish coated withfibronectin.

[0143] Examples of the transcription factors include a homeobox-typetranscription factor, Nkx2.5/Csx (SEQ ID NO:9, amino acid sequence; SEQID NO:10, nucleotide sequence); a zinc finger-type transcription factorbelonging to the GATA family, GATA4 (SEQ ID NO:11, amino acid sequence;SEQ ID NO:12, nucleotide sequence); transcription factors belonging tothe myocyte enhance factor-2 (MEF-2) family, MEF-2A (SEQ ID NO:13, aminoacid sequence; SEQ ID NO:14, nucleotide sequence), MEF-2B (SEQ ID NO:15,amino acid sequence; SEQ ID NO:16, nucleotide sequence), MEF-2C (SEQ IDNO:17, amino acid sequence; SEQ ID NO:18, nucleotide sequence) andMEF-2D (SEQ ID NO:19, amino acid sequence; SEQ ID NO:20, nucleotidesequence); transcription factors belonging to the basic helix loophelix-type transcription factors, dHAND (SEQ ID NO:21, amino acidsequence; SEQ ID NO:22, nucleotide sequence) and eHAND (SEQ ID NO:23,amino acid sequence; SEQ ID NO:24, nucleotide sequence); andtranscription factors belonging to the family of TEA-DNA binding-typetranscription factors, TEF-1 (SEQ ID NO:25, amino acid sequence; SEQ IDNO:26, nucleotide sequence), TEF-3 (SEQ ID NO:27, amino acid sequence;SEQ ID NO:28, nucleotide sequence) and TEF-5 (SEQ ID NO:29, amino acidsequence; SEQ ID NO:30, nucleotide sequence).

[0144] The cardiomyogenic differentiation of the cells having thepotential to differentiate into cardiomyocytes can be induced byintroducing DNA encoding one or combination of the above-describedfactors into the cells and expressing the DNA therein.

[0145] It is also possible to induce the cardiomyogenic differentiationof the cells having the potential to differentiate into cardiomyocytesby culturing them using a culture dish coated with an extracellularmatrix obtained from spontaneously beating cardiomyocytes, co-culturingwith spontaneously beating cardiomyocytes or adding a culturesupernatant of spontaneously beating cardiomyocytes.

[0146] Furthermore, a factor which induces differentiation ofcardiomyocytes which are obtained by the method described in 4 below(hereinafter referred to as “the cardiomyogenic differentiation-inducingfactor”) can also be used in inducing the cardiomyogenic differentiationof the cells having the potential to differentiate into cardiomyocytes.

[0147] 4. Methods for Obtaining Cardiomyogenic Differentiation-InducingFactors

[0148] A cardiomyogenic differentiation-inducing factor can be obtainedby adding various protease inhibitors to a culture supernatant ofspontaneously beating cardiomyocytes, followed by combinations oftreatments, such as dialysis, salting-out and chromatography.

[0149] Genes encoding such cardiomyogenic differentiation-inducingfactors can be obtained by determining partial amino acid sequences ofthese factors using a microsequencer followed by screening a cDNAlibrary prepared from the spontaneously beating cells using DNA probesdesigned based on the determined amino acid sequences.

[0150] 5. Therapeutic Agents for Cardiac Regeneration and TherapeuticAgents for Heart Diseases Comprising Cells having the Potential toDifferentiate into Cardiomyocytes

[0151] The cells having the potential to differentiate intocardiomyocytes according to the present invention can be used astherapeutic agents for cardiac regeneration or for heart diseases.

[0152] The heart diseases include myocardial infarction, ischemic heartdisease, congestive heart failure, arrhythmia, hypertrophiccardiomyopathy, dilated cardiomyopathy, myocarditis and valvulardisease.

[0153] The agents for cardiac regeneration contain the cells having thepotential to differentiate into cardiomyocytes of high purity whichcells have been proliferated in vitro according to the position and sizeof the damaged part of the heart. The preferred cells having thepotential to differentiate into cardiomyocytes are those which can beinduced to differentiate into various cells constituting the heart suchas endocardial endothelial cells, cushion cells, ventricularcardiomyocytes, atrial cardiomyocytes and sinus node cells.

[0154] The therapeutic agents can be prepared by purifying the cellshaving the potential to differentiate into cardiomyocytes from the bonemarrow fluid taken from myocardial infarction patients according to theabove-described density gradient centrifugation, the panning method (J.Immunol., 141(8): 2797-800 (1988)) or the FACS method (Int. Immunol.,275-83 (1998)) using the antibodies described below which specificallyrecognize the cells having the potential to differentiate intocardiomyocytes, or a method for constructing a reporter system using thepromoter of a gene specifically expressed in the cell having thepotential to differentiate into cardiomyocytes.

[0155] The therapeutic agents include cardiomyocytes derived from thecells having the potential to differentiate into cardiomyocytes usingthe myocardium-forming agent described below as well as the cells havingthe potential to differentiate into cardiomyocytes which are obtained byactivating the division potential of the bone marrow cells taken fromthe bone marrow of aged persons by utilizing the immortalization methoddescribed below.

[0156] The purity of the therapeutic agents prepared according to theabove methods can be tested by the FACS method combined with theantibodies which specifically recognize the cells having the potentialto differentiate into cardiomyocytes.

[0157] The therapeutic agents can be transported to the damaged parts bya method using a catheter or the like. For example, in the case ofischemic heart disease, the therapeutic agents are transported accordingto the following procedure. Since the cardiomyocytes damaged by ischemicheart disease exist downstream of vascular stricture, it is necessary tolocate the vascular stricture by coronary arteriography (IllustratedPathological Internal Medical Course Circulateory Organ, 1, MEDICALVIEW, 1993) prior to the injection of the above cells. Organic strictureis classified as concentric stricture, eccentric stricture or multiplemural asymmetry according to type of stricture, and eccentric strictureis further classified into two types, i.e. type I and type II. It isknown that the types of stricture are related to the course andprognosis of angina; for instance, eccentric stricture of type II andmultiple mural asymmetry are often observed in unstable angina which isliable to shift into myocardial infarction. In cases where blood vesselsare completely strictured, there is the possibility that the injectedcells can not reach the damaged parts. In such cases, the stricturedparts must be reopened by means of percutaneous transluminal coronaryangioplasty (PTCA), thrombolytic treatment or the like prior to the cellinjection. The type of the cells to be injected such as ventricular oratrial can be selected according to the position of the damagedcardiomyocytes. The insertion of a catheter can be performed by theSones method (Illustrated Pathological Internal Medical CourseCirculateory Organ, 1, MEDICAL VIEW, 1993) through the artery of theright upper arm or by the Jundkins method (Illustrated PathologicalInternal Medical Course Circulateory Organ, 1, MEDICAL VIEW, 1993)through the femural artery.

[0158] 6. Myocardium-Forming Agents

[0159] The myocardium-forming agents according to the present inventioncomprise, as an active ingredient, at least one cardiomyogenicdifferentiation-inducing factor selected from the group consisting of achromosomal DNA-demethylating agent, a factor which is expressed in thecardiogenesis region of a fetus, and a factor which acts ondifferentiation into cardiomyocytes in the cardiogenesis stage of afetus, and are capable of inducing the bone marrow-derived cells todifferentiate into cardiomyocytes.

[0160] Examples of the cardiomyogenic differentiation-inducing factorsinclude cytokines, vitamins, adhesion molecules and transcriptionfactors.

[0161] Any cytokine can be used, so long as it stimulates thecardiomyogenic differentiation of the cells having the potential todifferentiate into cardiomyocytes in the cardiogenesis stage.

[0162] For example, PDGF, FGF-8, endotherin 1 (ET1), Midkine and BoneMarrow Protein 4 (BMP4) can be used. Preferable examples of the PDGF,FGF8, ET1, Midkine, BMP4 include those the amino acid sequencesrepresented by SEQ ID NOS:3 and 5, the amino acid sequence representedby SEQ ID NO:64, the amino acid sequence represented by SEQ ID NO:66,the amino acid sequence represented by SEQ ID NO:68, and the amino acidsequence represented by SEQ ID NO:70, respectively. The cytokine can beused, e.g., at a concentration of 10 to 40 ng/ml.

[0163] Any vitamin can be used, so long as it stimulates thecardiomyogenic differentiation of the cells having the potential todifferentiate into cardiomyocytes in the cardiogenesis stage. Retinoicacid can be used, e.g., at a concentration of 10⁻⁹ M.

[0164] Any adhesion molecule can be used so far as it is expressed inthe cardiogenesis region in the cardiogenesis stage. Examples includegelatin, laminin, collagen, fibronectin and the like. For example, thecardiomyogenic differentiation of the cells having the potential todifferentiate into cardiomyocytes can be stimulated by culturing thecells in a culture dish coated with fibronectin.

[0165] Examples of the transcription factors include a homeobox-typetranscription factor, Nkx2.5/Csx (SEQ ID NO:9, amino acid sequence; SEQID NO:10, nucleotide sequence); a zinc finger-type transcription factorbelonging to the GATA family, GATA4 (SEQ ID NO:11, amino acid sequence;SEQ ID NO:12, nucleotide sequence); transcription factors belonging tothe myocyte enhancer factor-2 (MEF-2) family, MEF-2A (SEQ ID NO:13,amino acid sequence; SEQ ID NO:14, nucleotide sequence), MEF-2B (SEQ IDNO:15, amino acid sequence; SEQ ID NO:16, nucleotide acid sequence),MEF-2C (SEQ ID NO:17, amino acid sequence; SEQ ID NO:18, nucleotidesequence) and MED-2D (SEQ ID NO:19, amino acid sequence; SEQ ID NO:20,nucleotide sequence); transcription factors belonging to the basic helixloop helix-type transcription factors, dHAND (SEQ ID NO:21, amino acidsequence; SEQ ID NO:22, nucleotide sequence), eHAND (SEQ ID NO:23, aminoacid sequence; SEQ ID NO:24, nucleotide sequence) and MesP1 (SEQ IDNO:61, amino acid sequence; SEQ ID NO:62, nucleotide sequence); andtranscription factors belonging to the family of TEA-DNA binding-typetranscription factors, TEF-1 (SEQ ID NO:25, amino acid sequence; SEQ IDNO:26, nucleotide sequence), TEF-3 (SEQ ID NO:27, amino acid sequence;SEQ ID NO:28, nucleotide sequence) and TEF-5 (SEQ ID NO:29, amino acidsequence; SEQ ID NO:30, nucleotide sequence).

[0166] The myocardium-forming agents can contain, as a main component,either a gene encoding a cardiomyogenic differentiation-inducing factoror a protein which is a cardiomyogenic differentiation-inducing factoritself.

[0167] (1) Myocardium-Forming Agent Containing Gene as Main Component

[0168] Methods for preparing the myocardium-forming agents of thepresent invention which comprise, as a main component, a gene encoding acardiomyogenic differentiation-inducing factor are described below.

[0169] First, a DNA fragment or the full length cDNA of a gene encodinga cardiomyogenic differentiation-inducing factor is inserted downstreamof a promoter in a virus vector plasmid to construct a recombinant virusvector plasmid.

[0170] Then, the obtained recombinant virus vector plasmid is introducedinto a packaging cell which is suitable for the virus vector plasmid.

[0171] The recombinant virus vector plasmid lacks at least one of thegenes encoding the proteins necessary for the packaging of a virus. Asthe packaging cell, any cell can be used so far as it can supply theprotein encoded by the lacking gene. Suitable packaging cells includeHEK293 cell derived from human kidney and mouse fibroblast NIH3T3.

[0172] Examples of the proteins supplied by the packaging cells includeproteins, such as gag, pol and env, derived from mouse retroviruses forretrovirus vectors; proteins, such as gag, pol, env, vpr, vpu, vif, tat,rev and nef, derived from HIV viruses for lentivirus vectors; proteins,such as E1A and E1B, derived from adenoviruses for adenovirus vectors;and proteins, such as Rep(p5, p19, p40) and Vp(Cap), foradeno-associated viruses.

[0173] The virus vector plasmids that can be employed are those capableof producing a recombinant virus in the above packaging cells andcomprising a promoter at a position appropriate for the transcription ofa wild-type gene corresponding to the causative gene of a congenitalgenetic heart disease in cardiomyocytes.

[0174] Suitable virus vector plasmids include MFG (Proc. Natl. Acad.Sci. USA, 92: 6733-6737 (1995)), pBabePuro (Nucleic Acids Research, 18:3587-3596 (1990)), LL-CG, CL-CG, CS-CG and CLG (Journal of Virology, 72:8150-8157 (1998)) and pAdex1 (Nucleic Acids Res., 23: 3816-3812 (1995)).

[0175] Any promoter can be used as long as it can be expressed in humantissues. Examples of suitable promoters are the promoter of IE(immediate early) gene of cytomegalovirus (human CMV), SV40 earlypromoter, the promoter of a retrovirus, metallothionein promoter, heatshock protein promoter and SRα promoter. The enhancer of IE gene ofhuman CMV may be used in combination with the promoter. It is possibleto express the desired gene specifically in cardiomyocytes using apromoter of a gene specifically expressed in cardiomyocytes such asNkx2.5/Csx gene.

[0176] A recombinant virus vector can be produced by introducing theabove recombinant virus vector plasmid into the above packaging cell.Introduction of the virus vector plasmid into the packaging cell can becarried out, for example, by the calcium phosphate method (JapanesePublished Unexamined Patent Application No. 227075/90) or thelipofection method (Proc. Natl. Acad. Sci. USA, 84: 7413 (1987)).

[0177] The above recombinant virus vector can be formulated intomyocardium-forming agents by admixture with a carrier used inpharmaceutical compositions for gene therapy (Nature Genet., 8: 42(1994)). Any carrier can be used so long as it is usually used ininjections. Suitable carriers include distilled water, salt solutions ofsodium chloride or mixtures of sodium chloride and inorganic salts,solutions of mannitol, lactose, dextran, glucose, etc., solutions ofamino acids such as glycine and arginine, and mixtures of organic acidsolutions or salt solutions and a glucose solution. Injections may beprepared in the form of solutions, suspensions or dispersed solutionsaccording to conventional methods using the above carriers as well asauxiliaries, for example, osmotic pressure adjusting agents, pHadjusting agents, vegetable oils such as sesame oil and soybean oil,lecithin, and surfactants such as nonionic surfactants. If desired, theinjections may be prepared in the form of powdered or freeze-driedpreparations which are dissolved in a solvent before each use. Themyocardium-forming agents in the form of liquid preparations can be usedas such for gene therapy, and those in the form of solid preparationsare dissolved, immediately before use, in the above carriers which aresterilized if necessary. Administration of the myocardium-forming agentsis made locally using a catheter or the like so that the agents can beabsorbed into the myocardium of a patient.

[0178] The cells having the potential to differentiate intocardiomyocytes infected with the above recombinant virus vector in vitrocan also be formulated into the above myocardium-forming agents andadministered to a patient. Furthermore, the recombinant virus vector canbe directly administered to the diseased part of a patient.

[0179] (2) Myocardium-Forming Agent Containing Protein as Main Component

[0180] Methods for preparing the myocardium-forming agents of thepresent invention which contains as a main component, a protein which isa cardiomyogenic differentiation-inducing factor are described, below.

[0181] On the basis of the full length CDNA encoding a cardiomyogenicdifferentiation-inducing factor, if necessary, a DNA fragment having anappropriate length containing a region encoding the protein is prepared.

[0182] The prepared DNA fragment or the full length cDNA is inserteddownstream of a promoter in an expression vector to construct arecombinant expression vector for the protein.

[0183] Then, the recombinant expression vector is introduced into a hostcell suited for the expression vector.

[0184] Any cell can be used so long as it is capable of expressing thedesired gene products. Examples of the host cells include bacteriabelonging to the genus Escherichia, the genus Serratia, the genusCorynebacterium, the genus Brevibacterium, the genus Pseudomonas, thegenus Bacillus and the genus Microbacterium, yeasts belonging to thegenus Kluyveromyces, the genus Saccharomyces, the genusShizosaccharomyces, the genus Trichosporon and the genus Schwanniomyces,animal cells and insect cells.

[0185] The expression vectors that can be employed are those capable ofautonomous replication or integration into chromosome in the above hostcells and containing a promoter at a position suitable for thetranscription of a gene of a cardiomyogenic differentiation-inducingfactor.

[0186] When bacteria are used as the host cell, it is preferred that therecombinant expression vector for a gene encoding a cardiomyogenicdifferentiation-inducing factor is a recombinant vector which is capableof autonomous replication in the bacterial cell and which comprises apromoter, a ribosome binding sequence, a DNA encoding a protein whichcan induce cardiomyogenic differentiation, and a transcriptiontermination sequence. The vector can further comprise a gene regulatingthe promoter.

[0187] Examples of suitable expression vectors include pBTrp2, pBTac1and pBTac2 (manufactured by Boehringer Mannheim), pKK233-2 (manufacturedby Amersham Pharmacia Biotech), pSE280 (manufactured by Invitrogen),pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN),pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83),pKYP200 (Agricultural Biological Chemistry, 48: 669 (1984)), pLSA1(Agric. Biol. Chem., 53: 277 (1989)), pGEL1 (Proc. Natl. Acad. Sci. USA,82: 4306 (1985)), pBluescript II SK (−) (manufactured by Stratagene),pGEX (manufactured by Amersham Pharmacia Biotech), pET-3 (manufacturedby Novagen), pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094 and 5,160,735),and pSupex, pUB110, pTP5, pC194 and pEG400 (J. Bacteriol., 172: 2392(1990)).

[0188] It is preferred to use a plasmid in which the distance betweenthe Shine-Dalgarno sequence (ribosome binding sequence) and theinitiation codon is adjusted to a suitable length (e.g., 6-18 bases).

[0189] Any promoter can be used so long as it can be expressed in thehost cell. For example, promoters derived from Escherichia coli or aphage, such as trp promoter (P_(trp)), lac promoter (P_(lac)), P_(L)promoter, P_(R) promoter and T7 promoter, SPO1 promoter, SPO2 promoterand penP promoter can be used. Artificially modified promoters such as apromoter in which two Ptrp are combined in tandem (P_(trp)×2), tacpromoter, letI promoter (Gene, 44: 29 (1986)) and lacT7 promoter canalso be used.

[0190] The yield of the desired protein can be improved by replacing anucleotide in the nucleotide sequence of the protein-encoding region inthe gene of the cardiomyogenic differentiation-inducing factor of thepresent invention so as to make a codon most suitable for the expressionin a host cell.

[0191] The transcription termination sequence is not essential for theexpression of the gene encoding the cardiomyogenicdifferentiation-inducing factor of the present invention, but it ispreferred that the transcription termination sequence is locatedimmediately downstream of the structural gene.

[0192] Examples of suitable host cells are cells of microorganismsbelonging to the genus Escherichia, the genus Serratia, the genusCorynebacterium, the genus Brevibacterium, the genus Pseudomonas, thegenus Bacillus and the genus Microbacterium, specifically, Escherichiacoli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1,Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coliW1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coliNo. 49, Escherichia coli W3110, Escherichia coli NY49, Bacillussubtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes,Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticumATCC 14066, Corynebacterium glutamicum ATCC 13032, Corynebacteriumglutamicum ATCC 14067, Corynebacterium glutamicum ATCC 13869,Corynebacterium acetoacidophilum ATCC 13870, Microbacteriumammmoniaphilum ATCC 15354 and Pseudomonas sp. D-0110.

[0193] Introduction of the recombinant vector can be carried out by anyof the methods for introducing DNA into the above host cells, forexample, the method using calcium ion (Proc. Natl. Acad. Sci. USA, 69:2110 (1972)), the protoplast method (Japanese Published UnexaminedPatent Application No. 248394/88) and the methods described in Gene, 17:107 (1982) and Molecular & General Genetics, 168: 111 (1979).

[0194] When yeast is used as the host cell, YEp13 (ATCC 37115), YEp24(ATCC 37051), YCp50 (ATCC 37419), pHS19, pHS15, etc. can be used as theexpression vector.

[0195] Any promoter can be used, so long as it can be expressed in theyeast. Suitable promoters include PH05 promoter, PGK promoter, GAPpromoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shockprotein promoter, MFα 1 promoter and CUP 1 promoter.

[0196] Examples of suitable host cells include cells of Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Trichosporon pullulans and Schwanniomyces alluvius.

[0197] Introduction of the recombinant vector can be carried out by anyof the methods for introducing DNA into yeast cells, for example,electroporation (Methods. Enzymol, 194: 182 (1990)), the spheroplastmethod (Proc. Natl. Acad. Sci. USA, 75: 1929 (1978)) and the lithiumacetate method (J. Bacteriol., 153: 163 (1983), Proc. Natl. Acad. Sci.USA, 75: 1929 (1978)).

[0198] When an animal cell is used as the host cell, pcDNAI(manufactured by Invitorogen), pcDM8 (manufactured by Invitorogen),pAGE107 (Japanese Published Unexamined Patent Application No. 22979/91,Cytotechnology, 3: 133 (1990)), pAS3-3 (Japanese Published UnexaminedPatent Application No. 227075/90), pCDM8 (Nature, 329: 840 (1987)),pcDNAI/Amp (manufactured by Invitrogen), pREP4 (manufactured byInvitrogen), pAGE103 (J. Biochem. 101: 1307 (1987)), pAGE210, etc. canbe used as the expression vector.

[0199] As the promoter, any promoters capable of expression in animalcells can be used. Suitable promoters include the promoter of IE(immediate early) gene of cytomegalovinus (human CMV), SV40 earlypromoter, the promoter of a retrovirus, metallothionein promoter, heatshock protein promoter and SRα promoter. The enhancer of IE gene ofhuman CMV may be used in combination with the promoter.

[0200] Examples of suitable host cells are human Namalwa cell, monkeyCOS cell, Chinese hamster CHO cell and HBT5637 (Japanese PublishedUnexamined Patent Application No. 299/88).

[0201] Introduction of the recombinant vector can be carried out by anyof the methods for introducing DNA into animal cells, for example,electroporation method (Cytotechnology, 3: 133 (1990)), the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), and lipofection method (Proc. Natl. Acad. Sci. USA, 84: 7413(1987), Virology, 52: 456 (1973)). A transformant can be obtained andcultured according to the methods described in Japanese PublishedUnexamined Patent Application Nos. 227075/90 and 257891/90.

[0202] When an insect cell is used as the host cell, the protein can beexpressed using the methods descried in Baculovirus Expression Vectors,A Laboratory Manual, W. H. Freeman and Company, New York (1992), CurrentProtocols in Molecular Biology, Supplement 1-38 (1987-1997),Bio/Technology, 6: 47 (1988), etc.

[0203] Specifically, the recombinant gene transfection vector and abaculovirus are cotransfected into an insect cell to obtain arecombinant virus in the culture supernatant of the insect cell, andthen an insect cell is infected with the recombinant virus to expressthe protein.

[0204] Examples of the gene transfection vectors suitable for use inthis method are pVL1392, pVL1393 and pBlueBacIII (manufactured byInvitrogen).

[0205] Examples of the baculovirus include Autographa californicanuclear polyhedrosis virus with which an insect belonging to the familyBarathra is infected.

[0206] Examples of the insect cells include Sf9 and Sf21 (BaculovirusExpression Vectors, A Laboratory Manual, W. H. Freeman and Company, NewYork (1992)), which are ovary cells of Spodoptera frugiperda, and High 5(manufactured by Invitrogen), which is an ovary cell of Trichoplusia ni.

[0207] Cotransfection of the recombinant gene transfection vector andthe baculovirus into an insect cell for the preparation of therecombinant virus can be carried out by the calcium phosphate method(Japanese Published Unexamined Patent Application No. 227075/90), thelipofection method (Proc. Natl. Acad. Sci. USA, 84: 7413 (1987)), etc.

[0208] Expression of the gene can be carried out not only by directexpression but also by secretory production, fused protein expression,etc. according to the methods described in Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989) (hereinafter referred to as “Molecular Cloning, A LaboratoryManual, 2nd ed.”) etc.

[0209] When the gene is expressed in yeast, an animal cell or an insectcell, a glycoprotein or glycosylated protein can be obtained.

[0210] The protein as the cardiomyogenic differentiation-inducing factorcan be produced by culturing the transformant carrying the recombinantDNA containing the DNA encoding the protein as the cardiomyogenicdifferentiation-inducing factor in a medium, allowing the protein toaccumulate in the culture, and recovering the protein from the culture.

[0211] Culturing of the transformant for the production of the proteinas the cardiomyogenic differentiation-inducing faactor can be carriedout by conventional methods for culturing the host cell of thetransformant.

[0212] For the culturing of the transformant prepared using aprocaryotic cell such as E. coli or a eucaryotic cell such as yeast asthe host cell, any of natural media and synthetic media can be used, solong as it is a medium suitable for efficient culturing of thetransformant which contains a carbon source, a nitrogen source, aninorganic substance, etc. which can be assimilated by the host used.

[0213] Any carbon source can be used, so long as it can be assimilatedby the host. Examples of suitable carbon sources include carbohydratessuch as glucose, fructose, sucrose, molasses containing them, starch andstarch hydrolyzate; organic acids such as acetic acid and propionicacid; and alcohols such as ethanol and propanol.

[0214] Examples of the nitrogen sources include ammonia, ammonium saltsof inorganic or organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate and ammonium phosphate, and othernitrogen-containing compounds can be used as well as peptone, meatextract, yeast extract, corn steep liquor, casein hydrolyzate, soybeancake, soybean cake hydrolyzate, and various fermented cells and digestedproducts thereof.

[0215] Examples of the inorganic substances include potassiumdihydorgenphosphate, dipotassium hydrogenphosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate and calcium carbonate.

[0216] Culturing is usually carried out under aerobic conditions, forexample, by shaking culture or submerged spinner culture under aeration,at 15-40° C. for 16 hours to 7 days. The pH is maintained at 3.0-9.0during the culturing. The pH is adjusted using an organic or inorganicacid, an alkali solution, urea, calcium carbonate, ammonia, etc.

[0217] If necessary, antibiotics, such as ampicillin and tetracycline,can be added to the medium during the culturing.

[0218] When a microorganism transformed with an expression vectorcomprising an inducible promoter is cultured, an inducer may be added tothe medium, if necessary. For example, in the case of a microorganismtransformed with an expression vector containing lac promoter,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like can be added tothe medium; and in the case of a microorganism transformed with anexpression vector containing trp promoter, indoleacrylic acid (IAA) orthe like can be added.

[0219] For the culturing of the transformant prepared using an animalcell as the host cell, generally used media such as RPMI1640 medium (TheJournal of the American Medical Association, 199: 519 (1967)), Eagles'sMEM (Science, 122: 501 (1952)), Dulbecco's modified MEM (Virology, 8:396 (1959)) and 199 medium (Proceeding of the Society for the BiologicalMedicine, 73: 1 (1950)), media prepared by adding fetal calf serum tothese media, etc. can be used as the medium.

[0220] Culturing is usually carried out at pH 6-8 at 30-40° C. for 1-7days in the presence of 5% Co₂.

[0221] If necessary, antibiotics, such as kanamycin and penicillin, canbe added to the medium during the culturing.

[0222] For the culturing of the transformant prepared using an insectcell as the host cell, generally used media such as TNM-FH medium(manufactured by Pharmingen), Sf-900II SFM medium (manufactured by LifeTechnologies), ExCell 400 and ExCell 405 (manufactured by JRHBiosciences) and Grace's Insect Medium (Grace, T. C. C., Nature, 195:788 (1962)) can be used as the medium.

[0223] Culturing is usually carried out at pH 6-7 at 25-30° C. for 1-5days.

[0224] If necessary, antibiotics, such as gentamicin, can be added tothe medium during the culturing.

[0225] The protein as the cardiomyogenic differentiation-inducing factorcan be isolated and purified from the culture of the transformant byconventional methods for isolating and purifying proteins.

[0226] For example, when the protein as the cardiomyogenicdifferentiation-inducing factor is expressed in a soluble form in cells,the isolation and purification can be carried out in the followingmanner. After the completion of culturing, the cells are recovered fromthe culture by centrifugation and suspended in an aqueous buffer,followed by disruption using an ultrasonic disrupter, a French press, aManton Gaulin homogenizer, a Dyno Mill, etc. to obtain a cell-freeextract. The cell-free extract is centrifuged, and a purified proteinpreparation can be produced from the obtained supernatant using ordinarymeans for isolation and purification of proteins, for example,extraction with a solvent, salting-out with ammonium sulfate, etc.,desalting, precipitation with an organic solvent, anion exchangechromatography using resins such as diethylaminoethyl (DEAE)-Sepharoseand DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchangechromatography using resins such as S-Sepharose FF (manufactured byAmersham Pharmacia Biotech), hydrophobic chromatography using resinssuch as butyl Sepharose and phenyl Sepharose, gel filtration using amolecular sieve, affinity chromatography, chromatofocusing, andelectrophoresis such as isoelectric focusing, alone or in combination.

[0227] When the protein is expressed as an insoluble substance in cells,the cells are separated and disrupted, followed by centrifugation torecover the insoluble substance of the protein as a precipitatefraction.

[0228] The recovered insoluble substance of the protein is solubilizedwith a protein-denaturing agent. The solubilized protein solution isdiluted or dialyzed to lower the concentration of the protein-denaturingagent therein, thereby restoring the normal tertiary structure of theprotein, followed by the same isolation and purification steps asdescribed above to obtain a purified protein preparation.

[0229] When the protein as the cardiomyogenic differentiation-inducingfactor or its derivatives, such as a glycosylated protein, areextracellularly secreted, they can be recovered from the culturesupernatant. That is, the culture is treated by means such ascentrifugation and the obtained culture supernatant is subjected to thesame isolation and purification steps as mentioned above to obtain apurified protein preparation.

[0230] The thus obtained proteins include the proteins having the aminoacid sequences represented by SEQ ID NOS:5, 6, 10, 12, 14, 16, 18, 20,22, 24, 26, 28 and 30.

[0231] The proteins expressed by the above methods can also be producedby chemical synthetic methods such as the Fmoc method (thefluorenylmethyloxycarbonyl method) and the tBoc method (thet-butyloxycarbonyl method). Furthermore, the proteins can be synthesizedusing peptide synthesizers (for example, manufactured by AdvancedChemTech, Perkin-Elmer, Amersham Pharmacia Biotech, Protein TechnologyInstrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, etc.).

[0232] The protein which can induce cardiomyogenic differentiation canbe formulated into myocardium-forming agents and administered in thesame manner as in the above (1).

[0233] 7. Application to Therapy of Congenital Genetic Disease

[0234] In some of the diseases leading to heart failure, the deficiencyof an essential protein due to the mutation of a single gene causesheart failure. Examples of such diseases are familial hypertrophiccardiomyopathy, Fabri disease, QT elongation syndrome, Marfan syndrome,aortic stenosis, mitochondria cardiomyopathy and Duchenne musculardystrophy. These diseases are known to be caused by the abnormality inthe genes of myosin, troponin, tropomyosin, potential-dependent Nachannel, K channel, fibrin, elastin, mitochondria, dystrophin, etc.(Therapeutics, 30: 1302-1306 (1996)).

[0235] The method for treating a patient of the above disease includes amethod comprising acquiring the cells having the potential todifferentiate into cardiomyocytes of the present invention from apatient of the disease, introducing the wild type gene corresponding tothe causative gene of the disease into the cells, and transplanting thecells to the patient's heart. The normal gene is inserted into thevector for gene therapy described in the above 6(1), and then can beintroduced into the cells having the potential to differentiate intocardiomyocytes of the present invention using the vector for genetherapy described in the above 6(1).

[0236] 8. Methods for Obtaining Antibody which Specifically RecognizesSurface Antigen Specific for Cells having the Potential to Differentiateinto Cardiomyocytes

[0237] Methods for preparing antibodies which specifically recognizesurface antigens expressed in the cells having the potential todifferentiate into cardiomyocytes of the present invention are describedbelow.

[0238] The antibodies which recognize the surface antigens expressedspecifically in the cells having the potential to differentiate intocardiomyocytes of the present invention are useful in the purity testand purification of the cells required for applying the cells to thetherapy of heart diseases such as myocardial infarction.

[0239] In order to obtain the antibody, an antigen is administeredsubcutaneously, intravenously or intraperitoneally to a non-humanmammal, such as rabbit or goat, or 3 to 20-weeks-old rat, mouse orhamster together with an appropriate adjuvant, such as complete Freund'sadjuvant, aluminum hydroxide gel or pertussis vaccine. As the antigen,the cells having the potential to differentiate into cardiomyocytes ofthe present invention (3×10⁵ to 5×10⁵ cells/animal) or the cell membranefraction prepared from the cells (1-10 mg/animal) is used.

[0240] Administration of the antigen is repeated 3 to 10 times after thefirst administration at intervals of 1 to 2 weeks. On the 3rd to 7th dayafter each administration, a blood sample is collected from fundus oculiveniplex and the obtained serum is examined from reactivity to theantigen used for immunization according to enzyme immunoassay(Enzyme-Linked Immuno Adsorbent Assay (ELISA), Igaku Shoin (1976),Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)). Anon-human mammal whose serum shows a sufficient antibody titer againstthe antigen used for immunization is employed as a source of serum orantibody-producing cell.

[0241] The polyclonal antibody can be prepared by separation andpurification from the serum.

[0242] For the preparation of the monoclonal antibody, theantibody-producing cell and a myeloma cell derived from a non-humanmammal are fused to obtain hybridoma, and the hybridoma is cultured oradministered to an animal to cause ascites tumor. The monoclonalantibody can be prepared by separation and purification from theresulting culture or ascites.

[0243] Examples of the antibody-producing cells include spleen cells andantibody-producing cells in lymph nodes or peripheral blood, and amongthese, spleen cells are preferably used.

[0244] As the myeloma cells, mouse-derived cell lines are preferablyused. Examples of suitable cell lines are P3-X63Ag8-U1 (P3-U1) cell line(Current Topics in Microbiology and Immunology, 18: 1 (1978)), which is8-azaguanine-resistant mouse (BALB/c-derived) myeloma cell line,P3-NS1/1-Ag41(NS-1) line (European J. Immunology, 6: 511 (1976)),SP2/0-Ag14(SP-2) line (Nature, 276: 269 (1978)), P3-X63-Ag8653(653) line(J. Immunology, 123: 1548 (1979)) and P3-X63-Ag8(X63) line (Nature, 256:495 (1975)).

[0245] The hybridoma can be prepared in the following manner.

[0246] The antibody-producing cells and the myeloma cells are mixed andsuspended in HAT medium (a medium prepared by adding hypoxanthine,thymidine and aminopterin to a normal medium), followed by culturing for7-14 days. After the culturing, a portion of the culture supernatant issubjected to enzyme immunoassay to select cells which react with theantigen and do not react with the protein containing no antigen. Then,cloning is carried out by limiting dilution method, and cells showing ahigh and stable antibody titer according to enzyme immunoassay areselected as the monoclonal antibody-forming hybridomas.

[0247] Separation and purification of the polyclonal antibodies and themonoclonal antibodies can be carried out using means such ascentrifugation, ammonium sulfate precipitation, caprylic acidprecipitation, and chromatography using DEAE-Sepharose column, anionexchange column, protein A- or G-column or gel filtration column, aloneor in combination.

[0248] Sampling cells can be easily tested for expression of the surfaceantigen expressed in the cells having the potential to differentiateinto cardiomyocytes by comparing the reactivity of the thus obtainedantibody specifically which recognizes the surface antigen to the testcells with that to control cells such as hematopoietic stem cells andneural stem cells.

[0249] 9. Methods for Obtaining Surface Antigen Expressed in Cellshaving the Potential to Differentiate into Cardiomyocytes and geneEncoding the Surface Antigen

[0250] The genes encoding the surface antigens expressed specifically inthe cells having the potential to differentiate into cardiomyocytes canbe obtained by the cDNA subtraction method (Proc. Natl. Acad. Sci. USA,85: 5738-5742 (1988)) and the representational difference analysis(Nucleic Acids Research, 22: 5640-5648 (1994)), which are methods forobtaining genes showing different expression profiles between twosamples of different origins.

[0251] First, a cDNA library prepared from the cells having thepotential to differentiate into cardiomyocytes is subjected tosubtraction using mRNA obtained from control cells other than cellshaving the potential to differentiate into cardiomyocytes, e.g.,hematopoietic stem cells and neural stem cells. Then a subtracted cDNAlibrary with a high content of a gene specifically expressed in thecells having the potential to differentiate into cardiomyocytes isprepared, followed by nucleotide sequence analysis of inserted cDNA inthe subtracted cDNA library from the 5′ terminal side randomly to selectthose having the secretion signal sequence (random sequence analysis).The full length nucleotide sequences of the thus obtained cDNAs aredetermined to distinguish the proteins encoded by the cDNAs intosecretory proteins and membrane proteins.

[0252] In the above process, the signal sequence trap method can be usedinstead of the random sequence analysis (Science, 261: 600-603 (1993),Nature Biotechnology, 17: 487-490 (1999)). The signal sequence trapmethod is a method for selectively screening for genes having thesecretion signal sequence.

[0253] In order to efficiently obtain the specific surface antigens, itis preferred to prepare a signal sequence trap library from the cellshaving the potential to differentiate into cardiomyocytes using a vectorsuitable for subtraction and to subject the signal sequence trap libraryto subtraction using mRNA obtained from control cells such ashematopoietic stem cells and neural stem cells. The thus obtained DNAfragments containing the secretion signal sequence can be used as probesfor cloning the full length cDNAs.

[0254] The proteins encoded by the cDNAs can be distinguished intosecretory proteins and membrane proteins by determining the full lengthnucleotide sequences of the full length cDNAs.

[0255] When the obtained clone DNA, whether it is obtained by the randomsequence analysis or the signal sequence trap method, codes for amembrane protein, the specific antibody can be obtained by the abovemethod using the synthetic peptide prepared based on the amino acidsequence presumed from the nucleotide sequence as an antigen.

[0256] The membrane proteins encoded by the clones include receptors,which may act on the regulation of specific growth of cells having thepotential to differentiate into cardiomyocytes or their differentiationinto cardiomyocytes. The clone encoding such a receptor can be used inthe search for a ligand of the receptor. When the clone codes for asecretion protein, it can be used directly for the growth ordifferentiation of the cells having the potential to differentiate intocardiomyocytes.

[0257] 10. Methods for Screening for Growth Factor for Cells having thePotential to Differentiate into Cardiomyocytes and Factor Inducing theDifferentiation into Cardiomyocytes

[0258] Screening for a growth factor for the cells having the potentialto differentiate into cardiomyocytes and a factor inducing theirdifferentiation into cardiomyocytes can be carried out by culturing thecells having the potential to differentiate into cardiomyocytes in aserum-free medium in the presence of a test substance and evaluating thegrowth or the cardiomyogenic differentiation of the cells.

[0259] This screening method is applicable to a wide variety of testsubstances, for example, secretion proteins such as various cytokinesand growth factors, membrane-bound proteins such as cell adhesionmolecules, tissue extracts, synthetic peptides, synthetic compounds, andculture broths of microorganisms.

[0260] The growth capability can be evaluated by examining the colonyforming activity, the BrdU uptake, etc.

[0261] The colony forming activity can be examined by scattering thecells having the potential to differentiate into cardiomyocytes of thepresent invention at a low density.

[0262] The BrdU uptake can be examined by immunostaining using anantibody which specifically recognizes BrdU.

[0263] The cardiomyogenic differentiation can be evaluated according toa method using spontaneous beating as an indicator, a method using theexpression of a reporter gene introduced into the cells as an indicator,and the like.

[0264] The method using the expression of a reporter gene introducedinto the cells as an indicator is a method in which a vector DNAcomprising the promoter of a gene expressed specifically incardiomyocytes and a reporter gene is introduced into cells having thepotential to differentiate into cardiomyocytes and the expression of thereporter gene as an indicator is examined using the cells.

[0265] The reporter gene includes genes encoding GFP (gleen fluorescentprotein), luciferase or β-galactosidase, and the like.

[0266] The promoter of a gene expressed specifically in cardiomyocytesincludes cardiac troponin I (cTNI) (J. Biological Chemistry, 273:25371-25380 (1998)).

[0267] 11. Methods for Immortalizing Bone Marrow Cells having thePotential to Differentiate into Cardiomyocytes

[0268] When the therapeutic agent according to the present invention isadministered to cardiac patients, especially aged patients, it ispreferred that the proliferative activity of the cells having thepotential to differentiate into cardiomyocytes of the present inventionshould be potentiated without generating cancer.

[0269] The proliferative activity of the cells having the potential todifferentiate into cardiomyocytes can be increased without cancergeneration by expressing telomerase in the cells.

[0270] The methods for expressing telomerase in the cells having thepotential to differentiate into cardiomyocytes of the present inventioninclude: a method which comprises inserting TERT gene which is thecatalytic subunit of telomerase, specifically, the DNA represented bySEQ ID NO:32 into a retrovirus vector and introducing the resultingvector into the cells having the potential to differentiate intocardiomyocytes; a method which comprises administering a factor inducingthe expression of the TERT gene inherent in the cells having thepotential to differentiate into cardiomyocytes to the cells having thepotential to differentiate into cardiomyocytes; and a method whichcomprises introducing a vector containing DNA encoding a factor inducingthe expression of the TERT gene into the cells having the potential todifferentiate into cardiomyocytes.

[0271] The above-described factors inducing the expression of the TERTgene can be selected by introducing a vector DNA to which a reportergene such as GFP (green fluorescent protein), luciferase,P-galactosidase or the like has been inserted, into the cells having thepotential to differentiate into cardiomyocytes.

[0272] 12. Method of Separating Cells having the Potential toDifferentiate into Cardiomyocytes using Antibody

[0273] The method for obtaining cells in which a target surface antigenis expressed from extirpated various in vivo tissues includes a methodusing a flow cytometer having a sorting function and a method usingmagnetic beads.

[0274] The sorting function of a flow cytometer can be performed by thedroplet charge system, the cell capture system, etc. (Perfect Command ofFlow Cytometer, p.14-23, Shujunsha, 1999). In using each of thesesystems, the expression amount of an antigen can be quantitated byconverting the fluorescent intensity emitted from an antibody binding toa molecule expressed on the cell surface into an electric signal. Whenplural fluorescences are used in combination, the cells can be separatedusing plural surface antigens. Examples of the fluorescence include FITC(fluorescein insothiocyanate), PE (phycoerythrin), APC(Allo-phycocyanin), TR (TexasRed), Cy3, CyChrome, Red613, Red670, PerCP,TRI-Color, QuantumRed, etc. (Perfect Command of Flow Cytometer, p.3-13,Shujunsha, 1999).

[0275] The staining method includes a method in which cells arecentrifugally separated from extirpated various in vivo tissues such asbone marrow or umbilical blood, and the cells are stained directly withantibodies, and a method in which the cells are once cultured andproliferated in an appropriate medium and then stained with antibodies.

[0276] For staining, the target cells are first mixed with a primaryantibody, which recognizes a surface antigen, and incubated on ice for30 minutes to 1 hour. When the primary antibody is labeled with afluorescence, the cells are washed and then separated with a flowcytometer. When the primary antibody is not labeled with a fluorescence,the cells are washed and then a secondary antibody labeled with afluorescence having an activity of binding to the primary antibody ismixed with the cells having reacted with the primary antibody andincubated on ice again for 30 minutes to 1 hour. After washing, thecells stained with the primary and secondary antibodies are separatedwith a flow cytometer.

[0277] By the method using magnetic beads, cells expressing specifictarget surface antigen can be separated in a large amount. Although thismethod is inferior in the separation purity to the flow cytometer methodas described above, repeated purification ensures a sufficiently highcell purity.

[0278] After staining the cells with the primary antibody, the residualprimary antibody is eliminated. Then the cells are stained with thesecondary antibody bonded to the magnetic beads capable of binding tothe primary antibody. After washing away the residual secondaryantibody, the cells can be separated using a stand provided with amagnet. The materials and apparatus required in these operations areavailable from Dynal Biotech.

[0279] The magnetic bead method is also usable in eliminatingunnecessary cells from cell samples. The StemSep method marketed fromStem Cell Technologies Inc. (Vancouver, Canada) can be used to eliminatethese unnecessary cells more efficiently.

[0280] Examples of the antibodies to be used in the above-describedmethods include the antibodies acquired in the above 8, antibodies whichrecognize hematopoietic cell surface antigens, CD34, CD117, CD14, CD45,CD90, Sca-1, Ly6c or Ly6g, antibodies which recognize vascularendothelial cell surface antigens, Flk-1, CD31, CD105 or CD144, anantibody which recognizes a mesenchymal cell surface antigen, CD140,antibodies which recognize integrin surface antigens, CD49b, CD49d, CD29or CD41, and antibodies which recognize matrix receptors, CD54, CD102,CD106 or CD44. When these antibodies are used in combination, the targetcells can be obtained at a higher purity.

[0281] Specifically, in order to obtain CD34-negative, CD117-positive,CD144-negative and CD140-positive cells, CD34-positive cells andCD144-positive cells are eliminated from human bone marrow cells by, forexample, the above-described immune magnetic bead method and then aCD117-positive and CD140-positive cell fraction is recovered to separatethe target cells.

[0282] 13. Separation of Cardiomyocyte Precursor Cells usingMyocardium-Specific Gene Promoter Reporter Vector

[0283] In order to efficiently separate cardiomyocytes or cardiomyocyteprecursor cells derived from cells having the potential to differentiateinto cardiomyocytes, green fluorescent protein (GFP) of luminousAequorea can be used as a reporter gene for gene transfer.

[0284] Specifically, a vector is constructed by ligating the GFP gene tothe downstream of a promoter of a gene specifically expressed inmyocardium or a gene specifically expressed in the cells having thepotential to differentiate into cardiomyocytes obtained in the above 9.Then, the vector is introduced into the cells having the potential todifferentiate into cardiomyocytes. The cells introducing the reportervector are separated depending on, for example, tolerance to antibioticsfollowed by the induction of cardiomyogenic differentiation. Thedifferentiation-induced cells exhibit the expression of GFP and emitfluorescence. The cardiomyocytes and cardiomyocyte precursor cellsemitting the fluorescence can be easily separated using a flow cytometer(Perfect Command of Flow Cytometer, p.44-52, Shujunsha, 1999).

[0285] Examples of the promoter of the gene specifically expressed inmyocardium include MLC2v and troponin I.

[0286] Examples of the vector include the above-described plasmidvectors for animal cells, and adenovirus vectors.

[0287] 14. Induction of Differentiation of Cells having the Potential toDifferentiate into Cardiomyocytes into Various Cells

[0288] (1) Induction of Differentiation of Cells having the Potential toDifferentiate into Cardiomyocytes into Adipocytes

[0289] Examples of the method for inducing the differentiation of thecells having the potential to differentiate into cardiomyocytes intoadipocytes include a method wherein an activator of a nuclear receptor,PPARγ, is added to the medium to give a final concentration of 0.4 to 2μM. The activator of a nuclear receptor, PPARγ, includes compoundshaving a thiazolidione skeleton such as troglitazone, pioglitazone,rosiglitazone and the like.

[0290] The examples also include a method wherein the cells are culturedin a medium to which dexamethasone, methyl-isobutylxanthine, insulin andindomethacin have been added to a culture of cells confluently grownover a culture dish to give final concentrations of 1 μM, 0.5 mM, 0.01mg/ml and 0.2 mM, respectively.

[0291] (2) Induction of Differentiation of Cells having the Potential toDifferentiate into Cardiomyocytes into Chondrocytes

[0292] Examples of the method for inducing the differentiation of thecells having the potential to differentiate into cardiomyocytes intochondrocytes include a method wherein aggregates obtained bycentrifuging 1×10⁵ to 3×10⁵ cells are cultured in a medium containingTGFβ3 in a final concentration of 0.01 μg/ml.

[0293] (3) Induction of Differentiation of Cells having the Potential toDifferentiate into Cardiomyocytes into Osteoblasts

[0294] Examples of the method for inducing the differentiation of thecells having the potential to differentiate into cardiomyocytes intoosteoblasts include a method wherein the cells are cultured in a mediumcontaining dexamethasone, ascorbic acid-2-phosphate andβ-glycerophosphate in final concentrations of 0.1 μM, 0.05 mM and 10 mM,respectively.

[0295] 15. Purification of Stem Cell using Hoechst 33342

[0296] Hoechst 33342 is a DNA binding reagents which can stain viablecells. Since the majority of bone marrow cells are vigorously divided,they are stained markedly lightly but immature cells are stained darkly.It is known that this phenomenon becomes significant in cells havingimmature ability to exclude pigment by ABC (ATP binding cassette)transporter (H. Nakauchi, Protein, Nucleic Acid and Enzyme, 45: 13,2056-2062 (2000)).

[0297] Cells which are stained darkly with Hoechst 33342 can beseparated from the bone marrow by staining bone marrow cells withHoechst 33342 and then analyzing them by carrying out double staining ofa short wavelength and a long wavelength by applying UV laser usingFACS. Immature cells which do not incorporate Hoechst 33342 can befractionated as side population (Goodell, M. A. et al., J. Exp. Med.,183: 1797-1806 (1996),http://www.bcm.tmc.edu/genetherapy/goodell/new_site/index2.html).

BRIEF EXPLANATION OF THE DRAWINGS

[0298]FIG. 1 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using a biotinylated anti-mouse CD105 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0299]FIG. 2 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using a biotinylated anti-mouse Flk1 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0300]FIG. 3 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using an FITC-labeled anti-mouse CD31 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0301]FIG. 4 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using a biotinylated anti-mouse CD144 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0302]FIG. 5 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using an FITC-labeled anti-mouse CD34 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0303]FIG. 6 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using an FITC-labeled anti-mouse CD117(c-kit) antibodywhich was measured by a flow cytometer. The ordinate and abscissa showthe number of cells and the fluorescence intensity, respectively. Thearea painted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0304]FIG. 7 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using an FITC-labeled anti-mouse CD14 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0305]FIG. 8 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using an FITC-labeled anti-mouse CD45 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0306]FIG. 9 shows a result of the antibody reaction of KUM2 cell (A) orBMSC cell (B) using an FITC-labeled anti-mouse CD90 antibody which wasmeasured by a flow cytometer. The ordinate and abscissa show the numberof cells and the fluorescence intensity, respectively. The area paintedout with gray is a result of the antibody reaction, and the solid lineis a result of a negative control.

[0307]FIG. 10 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti- mouse Ly6A/E(Sca-1)antibody which was measured by a flow cytometer. The ordinate andabscissa show the number of cells and the fluorescence intensity,respectively. The area painted out with gray is a result of the antibodyreaction, and the solid line is a result of a negative control.

[0308]FIG. 11 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse Ly6c antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0309]FIG. 12 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse Ly6g antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0310]FIG. 13 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using a biotinylated anti-mouse CD140 antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0311]FIG. 14 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD49b antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0312]FIG. 15 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD49d antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0313]FIG. 16 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD29 antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0314]FIG. 17 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD54 antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0315]FIG. 18 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD102 antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0316]FIG. 19 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD106 antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0317]FIG. 20 shows a result of the antibody reaction of KUM2 cell (A)or BMSC cell (B) using an FITC-labeled anti-mouse CD44 antibody whichwas measured by a flow cytometer. The ordinate and abscissa show thenumber of cells and the fluorescence intensity, respectively. The areapainted out with gray is a result of the antibody reaction, and thesolid line is a result of a negative control.

[0318] The present invention are illustrated below based on thefollowing examples in more detail.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

[0319] Isolation and Culture of Bone Marrow Cells having the Potentialto Differentiate into Cardiomyocytes from Mouse Bone Marrow:

[0320] Ten 5-weeks-old C3H/He mice were anesthetized with ether andsacrificed by cervical dislocation. Each mouse was laid in half-lateralposition and sufficiently disinfected with 70% ethanol. The skin aroundthe femur was widely opened and the quadriceps femoris covering thefemur was excised with scissors. The femur was put out of the knee jointwith scissors and the muscle on the back side of the femur was removed.Then, the femur was put out of the hip joint with scissors and takenout. After the muscle on the femur was removed with scissors to exposethe whole femur, the femur was cut at both ends using scissors. A needle(23G, TERUMO) was attached to a 2.5 ml syringe and about 1.5 ml of IMDMcontaining 20% FCS was put into the syringe. The needle of the syringewas put into the femur from the cut end of the knee joint side and theculture medium was injected into the bone marrow, whereby bone marrowcells were pressed out of the bone into a test tube. The thus obtainedcell were cultured in IMDM supplemented with 20% FCS, 100 mg/mlpenicillin, 250 ng/ml streptomycin and 85 mg/ml amphotericin at 33° C.using a 5% CO₂-incubator. As a result of a series of passages, the cellswere homogenized into mesenchymal cells and hematopoietic cellsdisappeared.

[0321] After culturing for about 4 months under the above conditions,immortalized cells were selected and diluted to establish 192 cell linesrespectively derived from single cells (hereinafter referred to as bonemarrow-derived first passage immortalized cell lines). To each of theseclone-derived cell lines was added 5-aza-C at a final concentration of 3μM, and the cells were cultured for 24 hours. After culturing forfurther 2 weeks in IMDM, clones that produced spontaneously beatingcells were selected. Among the bone marrow-derived first passageimmortalized cell lines (192 cell lines), three cell lines were found tohave the potential to differentiate into cardiomyocytes. One of threecell lines is KUM2. Hereinafter, unless otherwise indicated, the bonemarrow cell KUM2 and mouse bone marrow-derived pluripotent stem cells(BMSC) described below were cultured in IMDM supplemented with 20% FCS,100 mg/ml penicillin, 250 ng/ml streptomycin and 85 mg/ml amphotericinat 33° C. using a 5% CO₂-incubator. When the KUM2 cells were exposed to5-aza-C having a final concentration of 3 μM for 24 hours, nonspecificdifferentiation into spontaneously beating cardiomyocytes was induced.However, the frequency is very low (one or less per 10⁷ cells).

[0322] However, cells surrounding the spontaneously beating cellsderived from the KUM2 cells were collected using a cloning syringe toobserve at least two cells of the mouse bone marrow-derived pluripotentstem cells (BMSC) having a high proliferation potentiality (FERMBP-7043) and cells differentiated into cardiomyocytes by proliferationunder limited times (hereinafter referred to as “cardiomyocyte precursorcells”). BMSC cells isolated by cloning syringe was cloned by selectingimmortalized cells in the course of multiple passage. It was observedthat the differentiation of the BMSC cells was induced at least 100times as efficient as the parent cell line, KUM2. And to thecardiomyocyte precursor cells, 5-aza-C was added, followed by culturingfor 24 hours, and further culturing in IMDM for 2-3 weeks, so that alarger number of spontaneously beating cells were efficiently obtained.The cardiomyocyte precursor cells showed mononuclear fibroblast-likemorphology under the proliferation conditions and expression ofmyocardial contractile proteins was hardly observed. However, inductionof final differentiation with 5-aza-C caused a remarkable change in themorphology of the cells.

[0323] About one week after the induction of differentiation, parts ofcells showed enlargement of cytoplasm and showed a ball-like orstick-like appearance. Such cells began spontaneously beating afterwardsbut spontaneous beating was still rare at this stage. Two weeks afterthe induction of differentiation, the cells began spontaneously beating.The spontaneously beating cells connected lengthwise with one another toform myotube-like structures. Three weeks after the induction ofdifferentiation, many cells were connected in a column andsimultaneously contracted. Four weeks after the induction ofdifferentiation, all of the directly connected cells on the culture dishshowed simultaneous contraction and formed a myocardial tissue-likestructure. The heart of a mouse contracts at a heart rate of 300-400 perminute. On the other hand, the cardiomyocytes differentiated from thecells derived from mouse adult bone marrow showed regular contraction ata rate of 120-250 per minute under the culture conditions.

EXAMPLE 2

[0324] Characteristics of the Cardiomyocytes Derived from Mouse BoneMarrow Cells:

[0325] The spontaneously beating cardiomyocyte-like cells produced fromthe bone marrow cells were examined for the characteristics ofcardiomyocytes.

[0326] Total RNAs were obtained from the bone marrow-derived firstpassage immortalized cell line, the mouse bone marrow-derivedpluripotent stem cells (BMSC), and the cardiomyocytes derived from thecardiomyocyte precursor cells, which were obtained in Example 1, usingTrizol Reagents (manufactured by GIBCO BRL). Then, first strand cDNAswere synthesized from the total RNAs as the substrates usingSuperscriptII reverse transcriptase (manufactured by GIBCO BRL).

[0327] In order to examine the expression of cardiomyocyte-specificgenes, quantitative PCR was carried out using the first strand cDNAs asthe substrates and using the synthetic DNAs having the nucleotidesequences represented by SEQ ID NOS:33 to 58. As thecardiomyocyte-specific genes, ANP and BNP, which are natriurecticpeptides, α-MHC and β-MHC, which are myosin heavy chains, α-skeletalactin and β-skeletal actin, which are actins, MLC-2a and MLC-2v, whichare myosin light chains, and Nkx2.5/Csx, GATA4, TEF-1, MEF-2C, MEF-2Dand MEF-2A, which are cardiomyocyte-specific transcription factors, wereemployed.

[0328] For the amplification of the above genes, the synthetic DNAshaving the nucleotide sequences shown in the following SEQ ID NOS wererespectively used: ANP, SEQ ID NOS:33 and 34; BNP, SEQ ID NOS:35 and 36;a-MHC, SEQ ID NOS:37 and 38; β-MHC, SEQ ID NOS:39 and 40; α-skeletalactin, SEQ ID NOS:41 and 42; β-skeletal actin, SEQ ID NOS:43 and 44;MLC-2a, SEQ ID NOS:45 and 46; MLC-2v, SEQ ID NOS:47 and 48; Nkx2.5/Csx,SEQ ID NOS:49 and 50; GATA4, SEQ ID NOS:51 and 52; TEF-1, SEQ ID NOS:53and 54; MEF-2C, SEQ ID NOS:55 and 56; MEF-2D, SEQ ID NOS:57 and 58; andMEF-2A, SEQ ID NOS:59 and 60.

[0329] In cardiomyocytes produced by induced differentiation in vivo,myocardial contractile proteins have different isoforms according to thedifference in stage, i.e., fetal period, new-born period or maturationperiod, or the difference in type, i.e., atrial or ventricular, so thatthe rate and energy efficiency of myocardial contraction may varyappropriately.

[0330] In the case of the bone marrow cells which differentiate intocardiomyocytes in vitro, a-skeletal actin was expressed at higher levelsthan a-cardiac actin in the expression pattern of isoforms; β-MHC wasexpressed at higher levels than α-MHC in the myosin heavy chain; andMLC-2v was expressed, whereas MLC-2a expression was not observed in themyosin light chain.

[0331] After the induction of differentiation of the bone marrow cellsinto cardiomyocytes in vitro, the expression of the natriureticpeptides, ANP and BNP, was observed. In view of the above expressionpattern of myocardial contractile proteins, it is considered that thebone marrow cells which differentiated into cardiomyocytes in vitro havea phenotype specific to fetal ventricular cardiomyocytes.

[0332] In the bone marrow cells which differentiated into cardiomyocytesin vitro, the expression of genes coding for Nkx2.5/Csx, GATA4, MEF-2A,MEF-2C, MEF-2D or TEF-1 was observed. The genes coding for thesetranscription factors were not expressed in the bone marrow-derivedfirst passage immortalized cell lines during proliferation. In the bonemarrow-derived cardiomyocyte precursor cells during proliferation, theexpression of genes coding for Nkx2.5/Csx, GATA4 or MEF-2C was observed.The expression of MEF-2A and MEF-2D was induced later with the inductionof cardiomyogenic differentiation.

[0333] The action potentials of the bone marrow cells whichdifferentiated into cardiomyocytes in vitro were recorded using glassmicroelectrodes. The cells were cultured in IMDM supplemented with 1.49mM CaCl₂, 4.23 mM KCl and 25 mM HEPES (pH 7.4), and the actionpotentials of the cells were measured at 25° C. under an invertedphase-contrast optic (Diaphoto-300, manufactured by Nikon). The glassmicroelectrodes were filled with 3M KCl and the electrode resistance wasset at 15-30 Ω in the glass microelectrodes. The membrane potentialswere measured with current clamp mode using MEZ-8300 (manufactured byNihon Kohden). The data were recorded on thermal recording papers usingRTA-1100M (manufactured by Nihon Kohden). As a result, it was found thatthe bone marrow cells which differentiated into cardiomyocytes in vitrowere classified into two types of action potentials: one is sinusnode-like action potential and the other is ventricular myocyte-likeaction potential. These two type cells of action potentials had thefollowing characteristics in common: (1) a long action potentialduration, (2) a relatively shallow resting potential, (3) pacemaker-likeslow depolarization of resting potential. The ventricular myocyte-likeaction potential showed the peak- and dome-like pattern having the phase1 action potential. The sinus node-like action potential showed theaction potential duration, diastolic membrane potential and actionpotential amplitude which are similar to those previously reported withthe action potentials of sinus node cells of rabbits and rats. Incomparison, the ventricular myocyte-like action potential had a tendencyto show a deep resting membrane potential and a high action potentialamplitude. During the 2-3 weeks after the induction of differentiation,the sinus node-like action potential was recorded for all the cells. Theventricular myocyte-like action potential was first recorded about 4weeks after the induction of differentiation and its incidence graduallyincreased with the passage of time.

EXAMPLE 3

[0334] Stimulation of Cardiomyogenic Differentiation using Cytokine:

[0335] The following experiment was conducted to investigate thestimulating effect of cytokines on the cardiomyogenic differentiation ofthe mouse bone marrow cells having the potential to differentiate intocardiomyocytes induced by 5-aza-C.

[0336] The mouse bone marrow-derived pluripotent stem cells (BMSC)having the potential to differentiate into cardiomyocytes were platedinto 60-mm culture dishes and 60 mm fibronectin-coated dishes (BectonDickinson) at a density of 2×10⁴ cells/ml and cultured at 33° C. in a 5%CO₂-incubator.

[0337] On the next day, 5-aza-C was added to each culture medium in afinal concentration of 3 μM, followed by culturing with the following 3different treatments, with addition of PDGF (culture dish A), both PDGFand retinoic acid (culture dish B) or without addition of any compound(culture dish C) (final concentration: PDGF, 10 ng/ml; retinoic acid,10⁻⁹ M).

[0338] On the next day, the medium was replaced with a fresh medium toremove 5-aza-C therefrom. Then, PDGF was added to the culture dish Auntil the final concentration of PDGF came to be 10 ng/ml, while PDGFand retinoic acid were added to the culture dish B until the finalconcentrations of PDGF and retinoic acid came to be 10 ng/ml and 10⁻⁹ M,respectively. Two and four days thereafter, the medium was replaced andthe PDGF or retinoic acid was further added.

[0339] Four weeks after the addition of the chemicals, the cellmorphology was observed with a phase-contrast microscope. As a result,about 30% of the cells in the culture dish containing 5-aza-C alonedifferentiated into myotubes, while about 40% of the cells in theculture dish containing PDGF and about 50% of the cells in the culturedish containing PDGF together with retinoic acid differentiated intomyotubes. In the three groups of the fibronectin-coated dishes, theratio of the cells differentiated into myotubes was about 10% higherthan in the three groups of the culture dishes.

[0340] RNAs were collected from the myotubes thus obtained. And genesexpressed in the myotubes were analyzed with quantitative PCR analysisusing the synthetic oligonucleotides represented by SEQ ID NOS:71 to 78.As a result, PDGF or retinoic acid promoted the expression of MyoD andfTnI genes relating to a skeletal muscle but not cTnI or ANPspecifically relating to a myocardium. Next, mouse bone marrow-derivedpluripotent stem cells (BMSC) having the potential to differentiate intocardiomyocytes were inoculated in a 60-mm culture dish at a density of2×10⁴ cells/ml and cultured using an incubator at 33° C. under 5% ofCO_(2.)

[0341] On the next day, 5-aza-C was added to the liquid culture mediumto give a final concentration of 3 μM. Furthermore, five treatmentsdiffering from each other were performed by adding FGF-8 to give a finalconcentration of 10 ng/ml (culture dish D); adding ET-1 to give a finalconcentration of 10 ng/ml (culture dish E); adding a midkine to give afinal concentration of 10 ng/ml (Culture dish F); adding BMP4 to give afinal concentration of 10 ng/ml (culture dish G); and adding no compound(culture dish H), followed by culturing.

[0342] On the next day, the medium was replaced by a fresh medium toeliminate 5-aza-C therefrom. Then, FGF-8 was added to the culture dish Dto give a final concentration of 10 ng/ml; ET-1 was added to the culturedish E to give a final concentration of 10 ng/ml; the midkine was addedto the culture dish F to give a final concentration of 10 ng/ml; andBMP4 was added to the culture dish G to give a final concentration of 10ng/ml, followed by culturing. Two and four days thereafter, the mediumwas replaced and the FGF-8, ET-1, midkine or BMP4 was further added.

[0343] Four weeks after the addition of 5-aza-C, the cell morphology wasobserved with a phase-contrast microscope. As a result, about 30% of thecells in the culture dish containing 5-aza-C alone differentiated intomyotubes, while about 50% of the cells in the culture dishes containingFGF-8, ET-1, midkine or BMP4 differentiated into myotubes respectively.

[0344] RNAs were collected from the myotubes thus obtained. And genesexpressed in the myotubes were analyzed with quantitative PCR using thesynthetic oligonucleotides represented by SEQ ID NOS:71 to 78. As aresult, the FGF-8, ET-1, midkine and BMP4 each individually promoted theexpression of cTnI and ANP gene which are myocardium-specific genes.

EXAMPLE 4

[0345] Induction of Differentiation of Bone Marrow-Derived Stem Cellsinto Cardiomyocytes using DMSO:

[0346] According to the method described in Example 1, mouse bonemarrow-derived pluripotent stem cells (BMSC) having the potential todifferentiate into cardiomyocytes were obtained and cultured for 24hours in the presence of 10 μM DMSO instead of 3 μM 5-aza-C. The mediumwas replaced with IMDM, followed by culturing for 6 weeks.

[0347] As a result, the stem cells were induced to differentiate intobeating cardiomyocytes. The produced cells expressed Nkx2.5/Csx andGATA4 genes and were found to be cardiomyocytes having the sameproperties as those obtained by the 5-aza-C treatment. This resultindicates that cardiomyogenic differentiation requires demethylation ofchromosomal DNA, which is a function common to 5-aza-C and DMSO.

EXAMPLE 5

[0348] Demonstration that Mouse Bone Marrow-Derived Pluripotent Cellshaving the Potential to Differentiate into Cardiomyocytes arePluripotent Stem Cells and Cardiomyocyte Precursor Cells:

[0349] It was demonstrated above that the beating cells differentiatedfrom the mouse bone marrow-derived pluripotent stem cell (BMSC) have theproperties of cardiomyocytes. In this example, a single cell markingexperiment was carried out to examine whether cardiomyocyte precursorcells are present in the mouse bone marrow-derived pluripotent stemcells (BMSC) having the potential to differentiate into cardiomyocytes,or whether more undifferentiated stem cells which can differentiate intonot only cardiomyocytes, but also, for example, adipocytes and othercell types are present.

[0350] Specifically, a GFP gene was inserted into a virus vector and thevector was transfected into a cell for labeling prior to induction ofdifferentiation, and the labeled cell was induced to differentiate toobserve what kind of cell is produced by differentiation.

[0351] First, retrovirus vector plasmid GAR3-GFP which expresses the GFPgene products and plasmid vector pCMV-Eco which expresses the Ecotropicgene products were treated according to the alkali neutralization methodand the PEG precipitation method described in Molecular Cloning, ALaboratory Manual, 2nd ed. to obtain DNAs of high purity.

[0352] One day before DNA transfection, 293 cells carrying the gag andpol genes which had reached confluence were passaged into a 10-cm dishby ⅕ dilution and cultured overnight at 37° C. in a 5% CO₂-incubator.

[0353] Transfection was carried out as follows.

[0354] GAR3-GFP retrovirus vector plasmid DNA (15 μg) and pCMV-Ecoplasmid vector DNA (5 μg) were dissolved in 0.5 ml of 250 mM CaCl₂ (pH6.95). The resulting solution was added dropwise to a 15 ml tubecontaining 0.5 ml of 2× BBS (50 mM BES(N,N-bis(2-hydroxyethl)-2-aminoethanesulfonic acid), 280 mM NaCl and 1.5mM Na₂HPO₄ (pH 6.95)) and the tube was allowed to stand at roomtemperature for 10 minutes. The resulting DNA solution was addeddropwise to the 293 cell culture prepared on the preceding day, followedby culturing at 37° C. in a 5% CO₂-incubator. On the next day, themedium was replaced with a fresh medium, followed by culturing at 37° C.in the 5% CO₂-incubator.

[0355] Two days after the medium replacement, the culture supernatantwas filtered through a 0.45 μm filter (manufactured by Millipore) torecover a solution containing the virus vector. The obtained solutionwas diluted to 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 10⁵ with IMDM.

[0356] The mouse bone marrow-derived pluripotent stem cells having thepotential to differentiate into cardiomyocytes into which the virusvector was to be introduced were plated into 6-well dishes at a densityof 2×10⁴ cells/well on the day before virus infection.

[0357] To the diluted virus vector solution, hexadimethine bromide(polybrene) (manufactured by Sigma) was added to give a finalconcentration of 8 μg/ml. After 2 ml of the culture supernatant of themouse bone marrow-derived pluripotent stem cells (BMSC) having thepotential to differentiate into cardiomyocytes was replaced with 2 ml ofthe virus solution, culturing was carried out at 33° C. in a 5%CO₂-incubator. Five hours later, the culture supernatant was replacedwith a fresh IMDM, followed by culturing at 33° C. in the 5%CO₂-incubator.

[0358] After culturing for 2 days, the cells were observed for GFPexpression by a fluorescence microscope to obtain cell populationscontaining one GFP-positive cell in 1000 cells.

[0359] The obtained cells were plated into 35 mm glass base dishes(manufactured by Asahi Techno Glass) at a density of 8×10³ cells/dishfollowed by culturing at 33° C. in a 5% CO₂-incubator.

[0360] On the next day, 5-aza-C (manufactured by Sigma), PDGF-BB(manufactured by Peprotech) and all trans retinoic acid (manufactured bySigma) were added to the dishes to give final concentrations of 3 μM, 10ng/ml and 10⁻⁹ M, respectively. Two days and four days after theaddition, the medium was replaced with a fresh medium and PDGF-BB(hereinafter referred to as “PDGF”) and all trans retinoic acid wereadded at the same concentrations as above.

[0361] Four weeks after, the cultures were observed under a fluorescencemicroscope to examine the mode of differentiation of the GFP-positivecells. As a result, the following three kinds of cell populations wereobserved; cell populations in which all the GFP-positive cells werecardiomyocytes; cell populations in which cardiomyocytes andundifferentiated stem cells were GFP-positive; and cell populations inwhich cardiomyocytes, adipocytes and undifferentiated stem cells wereGFP-positive. It has thus been found that differentiation isstochastically derived from pluripotent stem cells through myocardialstem cells and then cardiomyocyte precursor cells. This result alsoindicates that the mouse bone marrow cells having the potential todifferentiate into cardiomyocytes comprise pluripotent stem cells.

EXAMPLE 6

[0362] Promotion of Differentiation into Cardiomyocytes by ForcedExpression of Transcription Factors:

[0363] The following experiment was carried out to examine the effect ofthe forced expression of transcription factors relating tocardiomyogenic differentiation on the cardiomyogenic differentiation ofthe bone marrow-derived pluripotent stem cells (BMSC) having thepotential to differentiate into mouse cardiomyocytes.

[0364] That is, the Nkx2.5/Csx or GATA4 gene was introduced into thecells using a virus vector prior to induction of differentiation, andthen the cells were induced to differentiate to examine the efficiencyof cardiomyogenic differentiation.

[0365] In order to express the Nkx2.5/Csx, Nkx2.5/Csx was inserted intoretrovirus vector plasmid PCLNCX (manufactured by Imgenex) to preparepCLNC-Nkx2.5/Csx.

[0366] Furthermore, in order to express GATA4, GATA4 was inserted intoplasmid pCLPCX in which the G418-resistant gene portion in retrovirusvector plasmid pCLNCX (manufactured by Imgenex) had been replaced withpuromycin-resistant genes, to prepare pCLPC-GATA4. The retrovirus vectorplasmids pCLNC-Nkx2.5/Csx and pCLPC-GATA4 and plasmid vector pCMV-Eco(manufactured by Imgenex) which expresses the Ecotropic gene weretreated according to the alkali neutralization method and the PEGprecipitation method described in Molecular Cloning, A LaboratoryManual, 2nd ed., etc. to obtain DNAs having high purity.

[0367] One day before DNA transfection, 293 cells carrying the gag andpol gene which had reached confluence were passaged into a 10-cm dish by⅕ dilution followed by culturing overnight at 37° C. in a 5%CO₂-incubator.

[0368] Transfection was carried out as described below.

[0369] 15 μg of retrovirus vector DNA, pCLNC-Nkx2.5/Csx or pCLPC-GATA4,and 5 μg of plasmid vector, pCMV-Eco, were added and dissolved in 0.5 mlof 250 mM CaCl₂ (pH 6.95). The resulting solution was added dropwise toa 15 ml tube containing 0.5 ml of 2×BBS (50 mM BES(N,N-bis(2-hydroxyethl)-2-aminoethanesulfonic acid), 280 mM NaCl and 1.5mM Na₂HPO₄ (pH 6.95)) and the tube was allowed to stand at roomtemperature for 10 minutes. The resulting DNA solution was addeddropwise to the 293 cell culture prepared on the preceding day, followedby culturing at 37° C. in a 5% CO₂-incubator. On the next day, themedium was replaced with a fresh medium, followed by culturing at 37° C.in the 5% CO₂-incubator.

[0370] Two days after the medium replacement, the culture supernatantwas filtered through a 0.45 μm filter (manufactured by Millipore) torecover a solution containing the virus vector.

[0371] The mouse bone marrow-derived pluripotent stem cells (BMSC)having the potential to differentiate into cardiomyocytes into which thevirus vector was to be introduced were plated into 6-well dishes at adensity of 2×10⁴ cells/well on the day before virus infection.

[0372] To the obtained virus vector solution, hexadimethrine bromide(polybrene) (manufactured by Sigma) was added to give a finalconcentration of 8 μg/ml. The culture medium was replaced with theculture medium for the mouse bone marrow-derived pluripotent stem cells(BMSC) having the potential to differentiate into cardiomyocytes,followed by culturing at 33° C. in a 5% CO₂-incubator. Five hours later,the medium was replaced with a fresh IMDM, followed by culturing at 33°C. in the 5% CO₂-incubator, and further culturing for 2 days.

[0373] G418 was added to the cells infected with the virus produced bytransferring pCLNC-Nkx2.5 and pCMV-Eco to give a final concentration of300 μg/ml, followed by culturing for further 7 days.

[0374] Separately, puromycin was added to the cells infected with thevirus produced by transferring PCLPC-GATA4 and pCMV-Eco to give a finalconcentration of 300 ng/ml, followed by culturing for further 7 days.

[0375] During this period, both cells partly died and were detached fromthe dish. The surviving cells were suspended with trypsin followed byplating into new culture dishes.

[0376] The obtained stable transformants for expression of Nkx2.5/Csx orGATA4 were induced for differentiation by the method in the aboveExample 3, and thus the differentiation efficiency into cardiomyocyteswas examined.

[0377] The KNX2.5 forced expressing bone marrow cells (BMSC-KNx2.5)having the potential to differentiate into cardiomyocytes and the GATA4forced expressing bone marrow cells (BMSC-GATA4) having the potential todifferentiate into cardiomyocytes were plated into 60-mm culture dishesat a density of 2×10⁴ cells/ml, followed by culturing at 33° C. in a 5%CO₂-incubator. On the next day, 5-aza-C was added to each culture mediumto give a final concentration of 3 μM. After continuing the culturing at33° C. in a 5% CO₂-incubator for further 24 hours, the medium wasreplaced with a fresh medium to eliminate 5-aza-C, followed by culturingfor additional 4 weeks. When observed with a phase-contrast microscope,the number of myotube showed no large change caused by the forcedexpression of Nkx2.5/Csx or GATA4. Next, RNAs were collected from themyotubes thus obtained and genes expressed in the myotubes were analyzedwith quantitative PCR using the synthetic oligonucleotides representedby SEQ ID NOS:71 to 78. As a result, it was observed that the forcedexpression of Nkx2.5/Csx or GATA4 promoted the expression of cTnI andANP which are myocardium-specific genes.

[0378] To simultaneously express both of the Nkx2.5/Csx and GATA4 genesin bone marrow cells having the potential to differentiate intocardiomyocytes, a retrovirus vector plasmid pCLPC-GATA4 was treated asdescribed above and bone marrow cells (BMSC-KNX2.5) with the forcedexpression of Nkx2.5/Csx having the potential to differentiate intocardiomyocytes were infected with the recombinant virus thusconstructed. Next, puromycin was added to give a final concentration of300 ng/ml to obtain a drug-tolerant clone (BMSC-Nkx2.5-GATA4).

[0379] The Nkx2.5/Csx and GATA4 co-forced expressing bone marrow cells(BMSC-Nkx2.5-GATA4) having the potential to differentiate intocardiomyocytes were plated into a 60-mm culture dish at a density of2×10⁴ cells/ml, followed by culturing at 33° C. in a 5% CO₂-incubator.

[0380] On the next day, 5-aza-C was added to the culture medium to givea final concentration of 3 μM. After culturing at 33° C. in a 5%CO₂-incubator for further 24 hours, the medium was replaced with a freshmedium to eliminate 5-aza-C, followed by culturing for 4 weeks. Whenobserved with a phase-contrast microscope, the number f myotube showedno large change caused by the forced expression of the Nkx2.5/Csx andGATA4 genes. However, the number of beating cardiomyocyte was 50 timesor more elevated than bone marrow cells with no forced expression ofthese genes having the potential to differentiate into cardiomyocytes.Next, RNAs were collected from the myotubes thus obtained and genesexpressed in the myotubes were analyzed with quantitative PCR using thesynthetic oligonucleotides represented by SEQ ID NOS:71 to 78. As aresult, it was observed that the forced expression of Nkx2.5/Csx andGATA4 promoted the expression of cTnI and ANP which aremyocardium-specific genes.

EXAMPLE 7

[0381] Promotion of Differentiation into Cardiomyocytes by Combinationof the Forced Expression of Transcriptional Factors with Cytokines:

[0382] By combining the above-described transcriptional factors(Nkx2.5/Csx and GATA4) promoting the differentiation into cardiomyocyteswith cytokines (FGF-8, ET-1, midkine and BMP4), effects on thedifferentiation into cardiomyocytes were analyzed.

[0383] The Nkx2.5/Csx and GATA4 co-forced expressing bone marrow cells(BMSC-Nkx2.5-GATA4) having the potential to differentiate intocardiomyocytes were plated into a 60-mm culture dish at a density of2×10⁴ cells/ml and cultured at 33° C. in a 5% CO₂-incubator.

[0384] On the next day, 5-aza-C was added to the culture medium to givea final concentration of 3 μM. Furthermore, 5 treatments differing fromeach other were carried out by adding FGF-8 to give a finalconcentration of 10 ng/ml (culture dish I); adding ET-1 to give a finalconcentration of 10 ng/ml (culture dish J); adding midkine to give afinal concentration of 10 ng/ml (culture dish K); adding BMP4 to give afinal concentration of 10 ng/ml (culture dish L); and adding nothing(culture dish M), followed by culturing.

[0385] On the next day, the medium was replaced with a fresh medium toeliminate 5-aza-C. Then, FGF-8 was added to the culture dish I to give afinal concentration of 10 ng/ml; ET-1 was added to the culture dish J togive a final concentration of 10 ng/ml; midkine was added to the culturedish K to give a final concentration of 10 ng/ml; and BMP4 was added tothe culture dish L to give a final concentration of 10 ng/ml, followedby culturing. Two and four days thereafter, furthermore, the medium wasreplaced, and the FGF-8, ET-1, midkine or BMP4 was added.

[0386] Four weeks after the addition of 5-aza-C, the cell morphology wasobserved with a phase-contrast microscope. As a result, about 30% of thecells in the culture dish containing 5-aza-C alone were converted intomyotubes, while about 50% of the cells in the culture dishes containingFGF-8, ET-1, midkine or BMP4 differentiated into myotubes respectively.On the other hand, the addition of FGF-8, ET-1, midkine or BMP4 causedno increase in beating cardiomyocytes.

[0387] From the myotubes thus obtained, RNAs were collected and genesexpressed in the myotubes were subjected to quantitative PCR analysisusing the synthetic oligonucleotides represented by SEQ ID NOS:71 to 78.As a result, the FGF-8, ET-1, midkine and BMP4 did not further promotethe expression of cTnI and ANP which had been promoted by the forcedexpression of Nkx2.5/Csx and GATA4.

EXAMPLE 8

[0388] Transplantation of Mouse Having the Potential to Differentiateinto Cardiomyocytes into Heart:

[0389] In order to examine whether or not bone marrow cells having thepotential to differentiate into cardiomyocytes would differentiate intomyocardia and thus take into the heart, the GFP labeled bone marrowcells (BMSC-GFP) having the potential to differentiate intocardiomyocytes as prepared in Example 5 were employed as donor cells forthe transplantation into mouse. Specifically, the following procedurewas performed. The GFP-labeled BMSCs were transiently treated with5-aza-C for 24 hours, then suspended in PBS to give a concentration of1×10⁸ cells/ml and stored on ice until immediately before thetransplantation. It had been confirmed by 0.05% erythrosine-stainingthat BMSCs could survive at a ratio of about 95%.

[0390] On the other hand, the recipient C3H/He mice (available fromCharles River Japan) were anesthetized with ether, and the anesthesiawas maintained by intraperitoneally administering 30 mg of thiopentalusing a Terumo syringe (1 ml) manufactured by Terumo Corp. The legs ofeach mouse were fixed on a cork board with tape, and its upper jaw wasalso fixed on the cork board with rubber in such a manner that the neckleaned back. At this stage, electrocardiography electrodes were put intoboth upper limbs and right side lower limb to monitor theelectrocardiogram. Next, the cervix was incised about 1 cm along thetrachea using Mayo scissors (NONAKA RIKAKI CO., LTD, NK-174-14), thethyroid gland was stripped to the right and left sides using a babycotton swab manufactured by Hakujuji, and then muscles around thetrachea were incised using micro scissors (NONAKA RIKAKI CO., LTD,NY-334-08) to expose the trachea. Next, the trachea was incised in about1 mm width using a micro-feather (a surgical knife), a needle of SurflowFlash (22G) manufactured by Terumo deformed into J-shape was insertedinto the opening and taken out from the oral cavity, and then thesyringe of Surflow Flash (20G) was inserted into the trachea using theneedle as a guide. By connecting a respirator (MODEL SN-480-7,manufactured by SHINANO SEISAKUSHO) to the syringe, 100% oxygen wasflowed at a rate of 1 ml/minute to start artificial respiration with atidal volume of 1 ml and a respiration frequency of 120/minute. Sinceair leaks out from the guide needle-inserted opening, the skin aroundthe trachea was closed by covering the trachea using mosquito forceps(manufactured by NONAKA RIKAKI CO., LTD.). Next, a region of about 2 cmfrom the sternal pedicel toward the cervix was incised using Mayoscissors and then the sternum was incised about 2 cm from the sternalpedicel toward the cervix. Bleeding was stopped using a bipolar electricknife, and then a 30G needle (metal hub exchange needle N730)manufactured by GL Science was connected to the Terumo syringe (1 ml)manufactured by Terumo Corp and 0.1 ml of a solution prepared bysuspending the donor cells in PBS was injected into the apical region.Next, the sternum and the skin were closed using 4-0 ETHIBOND X761manufactured by ETHICON, and the skin of the cervix was closed using thesame suture. After confirmation of the turn up of spontaneousrespiration, the respirator was taken out, and an infant warmer washeated to 37° C. to wait vigilance of the animal therein. Also, theprocedure of this test was carried out using DESIGN FOR VISON 4.5×SURGICAL TELESCOPES.

[0391] Tissues were taken out from the mouse 77 days after thetransplantation, fixed with 10% formalin and embedded in paraffin. Theembedded tissues were sliced with a microtome into pieces of 6 μm inthickness and adhered to slide glasses which had been coated withpoly-L-lysine. After eliminating paraffin by immersing in 10% xylene,the samples were washed with ethanol and then immersed in 0.3% H₂O₂ for30 minutes, followed by a pretreatment for the antibody reaction.

[0392] Then, the samples were washed with PBS and blocked by reactingwith a 5% normal swine serum solution. After blocking, the samples werewashed with PBS and then subjected to the antibody reaction by allowingto stand at 4° C. overnight together with a mouse anti-GFP monoclonalantibody (manufactured by CLONTECH). After washing with PBS, the sampleswere allowed to react with a peroxidase-labeled dextran-bonded goatanti-mouse immunoglobulin antibody (manufactured by DACO) at roomtemperature for 30 minutes. After washing with PBS again, a coloringsolution (10 μg/ml 3,3′-diaminobenzidine (DAB) tetrahydrochloride, 0.01%H₂0₂, 0.05 M Tris-HCl (pH 6.7)) was added and allowed to react for about10 minutes. Then, the reaction mixture was washed with PBS to stop thereaction. Furthermore, the slide glasses were stained with methyl green.The part of continuous pieces were stained with hematoxylin/eosin toclarify the morphology of the tissue pieces.

[0393] As a result, GFP-positive cells were observed in thecardiomyocytes and the vascular endothelial cells.

[0394] Thus, it can be concluded that the transplanted mouse bone marrowcells had differentiated into the cardiomyocytes and the vascularendothelial cells.

EXAMPLE 9

[0395] Promotion of Differentiation into Cardiomyocytes by CulturedCardiomyocyte-Derived Factor:

[0396] As shown in Example 8, the bone marrow cells (BMSC) having thepotential to differentiate into cardiomyocytes were differentiated intothe cardiomyocytes when transplanted into the heart. This resultsuggests that cardiomyocytes per se expresses a factor inducing thedifferentiation of bone marrow cells into cardiomyocytes. To examinethis hypothesis, a mouse fetal heart was taken out from a C3H/He mouseon the day 16 of pregnancy and a primary culture cell line ofcardiomyocytes (hereinafter referred to as the “culturedcardiomyocytes”) was established in accordance with a publicly knownmethod (Development of Method for Studying Heart and Blood, ed. bySetsuro Ebashi, Gakkai Shuppan Senta, (1983)).

[0397] To examine whether or not a factor secreted from the culturedcardiomyocytes has an activity of promoting heart differentiation, 5×10⁶cultured cardiomyocytes were cultured in a culture dish for 72 hours.Next, the culture supernatant was filtered through a 0.45 μm filter(manufactured by Millipore). The culture supernatant thus filtered wasmixed with the equivalent amount of a medium to give a culture medium(hereinafter referred to as the “conditioned medium”) containing thefactor secreted from the cultured cardiomyocytes.

[0398] Bone marrow cells (BMSC) having the potential to differentiateinto cardiomyocytes or Nkx2.5 and GATA4 forced expressing bone marrowcells (BMSC-Nkx2.5-GATA4) having the potential to differentiation intocardiomyocytes were cultured 6-cm culture dishes at a density of 1×10⁵cells and then the medium was replaced with the conditioned medium. Atthis point, 5-aza-C was added to give a final concentration of 3 μM. Onthe next day, the medium was replaced with the fresh conditioned medium,followed by culturing for further 4 weeks. During this period, themedium was replaced with the fresh conditioned medium once 3 days. Thus,it was observed that myotubes derived from the bone marrow cell (BMSC)having the potential to differentiate into cardiomyocytes showed noincrease but the expression of the two myocardium-specific genes (ANPand cTnI) was promoted by the addition of the conditioned medium. Incase of the Nkx2.5 and GATA forced expressing bone marrow cells(BMSC-Nkx2.5-GATA4) having the potential to differentiate intocardiomyocytes, on the other hand, the myotubes showed no increase andthe expression of the two myocardium-specific genes (ANP and cTnI) waspromoted at the same level as in Nkx2.5 and GATA4 by the addition of theconditioned medium, showing no promoting effect.

[0399] Next, it was examined whether or not cardiomyocyte-expressingextracellular matrix (ECM) has an activity of promoting thedifferentiation into cardiomyocytes, culture dishes whereincardiomyocytes had been cultured were treated with 0.45% trypsin/EDTAfor about 30 minutes to eliminate the cardiomyocytes. Thus, culturedishes coated with the extracellular matrix of the culturedcardiomyocytes (hereinafter referred to as the “ECM-coated dishes”) wereprepared. Subsequently, bone marrow cells (BMSC) having the potential todifferentiate into cardiomyocytes or compulsively both Nkx2.5 and GATA 4genes-expressed bone marrow cells (BMSC-Nkx2.5-GATA4) having thepotential to differentiate into cardiomyocytes were cultured in these6-cm culture dishes at a density of 1×10⁵ cells and then 5-aza-C wasadded to give a final concentration of 3 μM. On the next day, the mediumwas replaced with a fresh medium to eliminate 5-aza-C and the culturewas continued for further 4 weeks. During this period, the medium wasreplaced with a fresh medium once 3 days. Thus, it was observed thatmyotubes derived from the bone marrow cells (BMSC) having the potentialto differentiate into cardiomyocytes showed no increase but theexpression of the two myocardium-specific genes (ANP and cTnI) waspromoted by the coated dish. In case of the compulsively both Nkx2.5 andGATA4 genes-expressed bone marrow cells (BMSC-Nkx2.5-GATA4) having thepotential to differentiate into cardiomyocytes, on the other hand, themyotubes showed no increase and the expression of the twomyocardium-specific genes (ANP and cTnI) was promoted at the same levelas in Nkx2.5 and GATA4 by the addition of the conditioned medium,showing no promoting effect.

[0400] Next, 2×10⁴ cultured cardiomyocytes were co-cultured togetherwith 8×10⁴ bone marrow cells (BMSC) having the potential todifferentiate into cardiomyocytes or 8×10⁴ compulsively both Nkx2.5 andGATA4 genes-expressed bone marrow cells (BMSC-Nkx2.5-GATA4) having thepotential to differentiate into cardiomyocytes in 6-cm culture dishes.To distinguish the cultured cardiomyocytes from the bone marrow cells,the two types of bone marrow cells (BMSC and BMSC-Nkx2.5-GATA4) werelabeled with GFP as in Example 5. On the next day after theco-culturing, 5-aza-C was added to give a final concentration of 3 μM.On the next day, the medium was replaced with a fresh medium toeliminate 5-aza-C, followed by culturing for further 4 weeks. Duringthis period, the medium was replaced with a fresh medium once 3 day. Asa result, beating cardiomyocytes were increased about 10 times or morethan the case wherein BMSC or BMSC-Nkx2.5-GATA4 were cultured alone.Thus, it was found that the efficiency of the differentiation intocardiomyocytes can be elevated 500 times or more by combining the forcedexpression of the Nkx2.5 and GATA4 genes with the co-culturing withcardiomyocytes.

EXAMPLE 10

[0401] Analysis of Surface Antigens of KUM2 Cells and BMSCs:

[0402] Surface antigens of KUM2 cells and BMSCs were analyzed to clearlydifferentiate KUM2 cells from BMSCs and develop a method for efficientlyisolating and purifying cells having the potentiality of formingmyocardium from bone marrow.

[0403] The surface antigens employed in the analysis included 20antigens, i.e., CD105, Flk-1, CD31 and CD144 known as surface antigensof vascular endothelial cells, CD34, CD117(c-kit), CD14, CD45, CD90,Ly6A/E(Sca-1), Ly6c and ly6g known as surface antigens in hematopoieticcells, CD140 known as surface antigens of mesenchymal cells, integrinsCD49b, CD49d and CD29 and matrix receptors CD54, CD102, CD106 and CD44.

[0404] First, 1×10⁴ KUM2 cells or 1×10⁴ BMSC cells were pipetted into a96-well U-shaped plate. An anti-mouse CD105 antibody (manufactured byPharmingen), which had been biotin-labeled by a publicly known method(Enzyme Antibody Technique, Gakusai Kikaku (1985)), was added to abuffer for FACS (1% BSA-PBS, 0.02% EDTA, 0.05% NaN₃, pH 7.4), then addedto the wells and allowed to react on ice for 30 minutes. As a negativecontrol, rat IgG2a, K-purified antibody (manufactured by Pharmingen) wasused. After washing with the buffer twice, 20 μl of streptoavidin-PE(manufactured by Nippon Becton Dickinson) was added. Then the mixturewas allowed to react in the dark on ice for 30 minutes, washed with thebuffer thrice and suspended in 500 μl of the buffer. The fluorescenceintensity was measured with a flow cytometer and it was examined whetheror not the fluorescence intensity was increased by adding the antibody.The results are shown in FIG. 1. As a result, it was found that the KUM2cells and the BMSC cells were both CD105-negative.

[0405] Regarding the occurrence of the expression of the Flk-1 antigen,an antibody reaction was carried out in the manner similar to thatdescribed above, using a biotinylated anti-mouse Flk-1 antibody(PM-28181D, manufactured by Pharmingen), followed by measurement with aflow cytometer. The results are shown in FIG. 2. As a result, it wasfound that the KUM2 cells and the BMSC cells were both Flk-1-negative.

[0406] Regarding the occurrence of the expression of the CD31 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD31 antibody (PM-01954D, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 3. As aresult, it was found that the KUM2 cells and the BMSC cells were bothCD31-negative.

[0407] Regarding the occurrence of the expression of the CD144 antigen,an antibody reaction was carried out using a biotinylated anti-mouseCD144 antibody (PM-28091D, manufactured by Pharmingen) followed bymeasurement with a flow cytometer. The results are shown in FIG. 4. As aresult, it was found that the KUM2 cells were CD144-negative, while theBMSC cells were CD144-weak positive.

[0408] Regarding the occurrence of the expression of the CD34 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD34 antibody (PM-09434D, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 5. As aresult, it was found that the KUM2 cells were CD34-negative, while theBMSC cells were a mixture of CD34-positive cells and CD34-negativecells.

[0409] Regarding the occurrence of the expression of the CD117(c-kit)antigen, an antibody reaction was carried out using an FITC-labeledanti-mouse CD117 antibody (PM-01904D, manufactured by Pharmingen),followed by measurement with a flow cytometer. The results are shown inFIG. 6. As a result, it was found that the KUM2 cells wereCD117-negative, while the BMSC cells were CD117-positive.

[0410] Regarding the occurrence of the expression of the CD14 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD14 antibody (PM-09474, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 7. As aresult, it was found that the KUM2 cells were CD14-positive, while theBMSC cells were CD14-negative.

[0411] Regarding the occurrence of the expression of the CD45 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD45 antibody (PM-01114, manufactured by Pharmingen), followed by themeasurement with a flow cytometer. The results are shown in FIG. 8. As aresult, it was found that the KUM2 cells and the BMSC cells were bothCD45-negative.

[0412] Regarding the occurrence of the expression of the CD90 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD90 antibody (PM-22214, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 9. As aresult, it was found that the KUM2 cells and the BMSC cells were bothCD90-negative.

[0413] Regarding the occurrence of the expression of the Ly6A/E(Sca-1)antigen, an antibody reaction was carried out using an FITC-labeledanti-mouse Ly6A/E(Sca-1) antibody (PM-01164A, manufactured byPharmingen), followed by measurement with a flow cytometer. The resultsare shown in FIG. 10. As a result, it was found that the KUM2 cells andthe BMSC cells were both Ly6A/E(Sca-1)-positive.

[0414] Regarding the occurrence of the expression of the Ly6c antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseLy6c antibody (PM-01152, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 11. Asa result, it was found that the KUM2 cells and the BMSC cells were bothLy6c-positive.

[0415] Regarding the occurrence of the expression of the Ly6g antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseLy6g antibody (PM-01214, manufactured by Pharmingen), followed by themeasurement with a flow cytometer. The results are shown in FIG. 12. Asa result, it was found that the KUM2 cells and the BMSC cells were bothLy6g-negative.

[0416] Regarding the occurrence of the expression of the CD140 antigen,an antibody reaction was carried out using a biotinylated anti-mouseCD140 antibody (PM-28011A, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 13. Asa result, it was found that the KUM2 cells and the BMSC cells were bothCD140-positive.

[0417] Regarding the occurrence of the expression of the CD49b antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD49b antibody (PM-09794, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 14. Asa result, it was found that the KUM2 cells were CD49b-positive, whilethe BMSC cells were CD49b-negative.

[0418] Regarding the occurrence of the expression of the CD49d antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD49d antibody (PM-01274, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 15. Asa result, it was found that the KUM2 cells and the BMSC cells were bothCD49d-negative.

[0419] Regarding the occurrence of the expression of the CD29 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD29 antibody (PM-22634, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 16. Asa result, it was found that the KUM2 cells and the BMSC cells were bothCD29-positive.

[0420] Regarding the occurrence of the expression of the CD54 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD54 antibody (PM-01544, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 17. Asa result, it was found that the KUM2 cells were CD54-positive, while theBMSC cells were CD54-negative.

[0421] Regarding the occurrence of the expression of the CD102 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD102 antibody (PM-01804, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 18. Asa result, it was found that the KUM2 cells and the BMSC cells were bothCD102-negative.

[0422] Regarding the occurrence of the expression of the CD106 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD106 antibody (PM-01814 manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 19. Asa result, it was found that the KUM2 cells were CD106-positive, whilethe BMSC cells were CD106-negative.

[0423] Regarding the occurrence of the expression of the CD44 antigen,an antibody reaction was carried out using an FITC-labeled anti-mouseCD44 antibody (PM-28154, manufactured by Pharmingen), followed bymeasurement with a flow cytometer. The results are shown in FIG. 20. Asa result, it was found that the KUM2 cells and the BMSC cells were bothCD44-positive.

[0424] Table 1 shows the summarized analytical data obtained using theflow cytometer. TABLE 1 KUM2 BMSC Hemato CD34 − ±*¹ CD117 (c-kit) − +CD14 + − CD45 − − CD90(Thyl) − − Ly-6a/e(Scal) + + Ly6c + + Ly6g − −Endothelial Flk-1 − − CD31 − − CD105 − − CD144 − +*² Mesenchyaml CD140(PDGFR) + + Integrin CD49b(α2) + − CD49b(α4) − − CD29(β1) + + MatrixCD54(ICAM-1) + − CD102(ICAM-2) − − CD106(VCAM-1) + − CD44(Hyaluronate) ++

EXAMPLE 11

[0425] Concentration of Differentiation Precursor Cells Using MouseMLC2v Promoter:

[0426] In order to efficiently obtain cells having the potential todifferentiate into myocardium from mouse bone marrow-derived cellshaving the potential to differentiate into cardiomyocytes, a promoterexpression system of a mouse MLC2v (myosin light chain-2v) gene showingcardiomyocyte-specific expression was constructed. Specifically, an EGFPgene (manufactured by CLONTECH) was ligated to the downstream of thepromoter sequence of the mouse MLC2v gene followed by constructing apMLC-2-EGFP plasmid containing the expression unit ofneomycin-resistance gene. DNA of this plasmid was obtained by the alkalineutralization method described in Molecular Cloning, A LaboratoryManual, 2nd ed. etc.

[0427] 2 μg of the above-described DNA was introduced usingLIPOFECTAMINE (manufactured by LIFE TECHNOLOGY) into KUM2 cells, whichhad been cultured in a 6-well plate to give 1×10⁵ cells. Detailedprocedure was carried out in accordance with the manufacturer'sinstructions. Forty-eight hours after the gene transfection, G418(manufactured by Sigma) was added to give a final concentration of 1mg/ml followed by selecting survived cells which were transfected by thegene.

[0428] On the 14th day after the gene introduction, 5-aza-C was added togive a final concentration of 3 μM, and 24 hours thereafter, the mediumwas replaced and the differentiation was induced. From the day 3 afterthe induction of the differentiation, GFP-positive cells were observed.On the day 4 after the induction of the differentiation, GFP-positivecells were exclusively selected from among 1×10⁴ cells using an FACSCaliber (manufactured by Becton Dickinson) and the culturing was furthercontinued. As a result, 90% or more cells had differentiated into cellshaving a myotube-like structure, which indicates that cells withdifferentiation potency could be efficiently concentrated. Aftercollecting by FACS, these GFP-positive cells were transplanted inaccordance with the method of Example 10. As a result, these cellsdifferentiated not into hemoendothelium but specifically into muscletissues such as skeletal muscle and myocardium.

EXAMPLE 12

[0429] INDUCTION of Adipocytes from Mouse Bone Marrow-Derived Cellshaving the Potential to Differentiate into Cardiomyocytes:

[0430] Bone marrow cells (BMSC) having the potential to differentiateinto cardiomyocytes can be induced to differentiate not only intocardiomyocytes but also into adipocytes. To control the differentiationinto adipocytes, the conditions for the induction of the differentiationwere examined. First, the expression of PPAR-y receptors was analyzed bythe quantitative PCR method. As a result, it was found that PPAR-γ1receptor was expressed but PPARγ2 receptor was not expressed in theBMSCs. Subsequently, PPAR-γ receptor agonists, pioglitazone andtroglitazone, were added at various concentrations to bone marrow cells(BMSC) having the potential to differentiate into cardiomyocytes. As aresult, the differentiation into adipocytes was promoted, depending onthe concentration, and about 50% and 100% of the BMSCs differentiatedinto adipocytes respectively at 0.4 μM and 2 μM.

EXAMPLE 13

[0431] Induction of Differentiation into Neurocytes, Hapatocytes andCardiomyocytes of Mouse Bone Marrow-Derived Cells having the Potentialto Differentiate into Cardiomyocytes by Transplantation intoBlastocysts:

[0432] In order to obtain stable transformants of GFP-labeled bonemarrow cells (BMSC) having the potential to differentiate intocardiomyocytes, gene transfection was first performed in the followingmanner.

[0433] GFP was introduced into a retrovirus vector plasmid pCLNCX(manufactured by Imgenex) to prepare PCLNC-GFP. The retrovirus vectorplasmid PCLNC-GFP and a pCMV-Eco plasmid vector (manufactured byImgenex) capable of expressing an ecotropic gene were treated by thealkali neutralization method and the PEG precipitation method describedin Molecular Cloning, A Laboratory Manual, 2nd ed. etc. to obtain DNAsof high purity.

[0434] A day before the transfection of these DNAs, 293 cells carryinggag and pol genes, which had become confluent were subculured into a 10cm dish by a dilution ratio of ⅕ and cultured at 37° C. in a 5%CO₂-incubator.

[0435] The transfection was carried out in the following manner.

[0436] In 0.5 ml of 250 mM CaCl₂ (pH 6.95), 5 μg of the pCLNC-GFPretrovirus vector plasmid DNA and 5 μg of the pCMV-Eco plasmid vectorDNA were dissolved. The solution thus obtained was dropped into 0.5 mlof 2× BBS (50 mM BES (N,N-bis(2-hydroxyethl)-2-aminoethanesulfonciacid), 280 mM NaCl, 1.5 mM Na₂HPO₄ (pH 6.95)) in a 15 ml tube andallowed to stand at room temperature for 10 minutes. Subsequently, theDNA solution was dropped into the medium of the 293 cells prepared onthe previous day and cultured at 37° C. in a 5% CO₂-incubator. On thenext day, the medium was replaced and culture was further continued at37° C. in a 5% CO₂-incubator.

[0437] Two days after the replacement of the medium, the culturesupernatant was filtered through a 0.45 μm filter (manufactured byMillipore) and a solution containing the virus vector was collected.

[0438] The mouse bone marrow cells (BMSC) having the potential todifferentiate into cardiomyocytes, into which the virus vector wasintroduced, were plated into a 6-well dish at a density of 2×10⁴cells/well on the previous day of the infection with the virus.

[0439] Hexadimethrine bromide(polybrene) (manufactured by Sigma) wasadded to the virus vector-containing solution obtained above to give afinal concentration of 8 μg/ml. After replacing by the medium of themouse bone marrow cells (BMSC) having the potential to differentiateinto cardiomyocytes, followed by culturing at 33° C. in a 5%CO₂-incubator. Five hours thereafter, the medium was replaced with freshIMDM, followed by further culturing at 33° C. in a 5% CO₂-incubator.

[0440] After culturing for two days, G418 was added until the finalconcentration of G418 came to be 300 μg/ml, followed by furtherculturing for further 7 days. During this period, a part of the cellsdied to be suspended. The surviving cells were suspended with trypsinand scattered in a fresh culture dish.

[0441] The obtained GFP-labeled bone marrow-derived cells having thepotential to differentiate into cardiomyocytes were grown in a 6-cmculture dish. After eliminating the medium, 0.5 ml of 0.25% trypsin EDTAwas added and the treatment was carried out for 1 minute. Then, 1.5 mlof a fresh medium was added and the cells were suspended. After addingferal bovine serum (manufactured by Lexicon Genetics) and mixing, thecell suspension was poured into mouse blastocyst. The mouse balstocystswere obtained by spontaneously mating female C57B1/6J mice subjected tohyper-ovulation with male mice of the same line, taking out the uterus 4days thereafter, and perfusing the inside of the uterus with M15 medium.After allowing to stand at 37° C. under 5% CO₂ until the balstocystcavities sufficiently dilated, the balstocyst were transferred into M15medium containing 20 mM HEPES which was cooled to about 4° C. Then, 10to 15 BMSCs were microscopically injected into each balstocyst cavitywhile observing under an =inverted microscope (manufactured by Nikon)provided with a microinjector (manufactured by Narumo Kagaku) and amicromanipulator (manufactured by Narumo Kagaku). After allowing tostand at 37° C. under 5% CO₂ until the balstocyst cavities sufficientlydilated, the blastocysts were transplanted into the oviducal side of theuterus of female MCH mice with pseudopregnancy, followed byimplantation. The female MCH mice with pseudopregnancy were prepared bymating with vasoligated male MCH mice aged 10 weeks or more on 17:00three days before the transplantation at the ratio of 1:1. On 9:00 onthe next morning, vaginal plugs were confirmed, and two days thereafter,the female mice were used for the above-described purpose.

[0442] The mice thus born were sacrificed and organs were extirpated forobserving the expression of GFP. As a result, the expression of GFP wasobserved in the brain and the liver, which suggested that the BMSCs haddifferentiated into the nerve system and the liver. Genomic DNA wasobtained from the heart taken out from another individual and subjectedto PCR using the primers of SEQ ID NOS:79 and 80. As a result, it wasconfirmed that BMSCs were also incorporated into the heart. Theseresults indicate that BMSCs have a totipotency of differentiating intoall of the three germ layers of nerve, heart and liver.

EXAMPLE 14

[0443] Telomerase Activity in Mouse Bone Marrow Cells having thePotential to Differentiate into Cardiomyocytes:

[0444] The mouse bone marrow cells having the potential to differentiateinto cardiomyocytes were examined for telomerase activity by theTelomeric Repeat Amplification Protocol (TRAP) method (TRAPezeTelomerase Detection kit, manufactured by Oncor). The measurement of thetelomerase activity was carried out as described below according to, inprinciple, the manufacture's instructions. The mouse bone marrow cellshaving the potential to differentiate into cardiomyocytes which had beencultured in a 6-cm culture dish (about 106 cells) were washed with PBS,followed by addition of 200 μl of 1× CHAPS solution. After being allowedto stand on ice for 30 minutes, the cells were recovered together withthe solution to a 1.5 ml centrifuge tube and centrifuged at 14000 rpmfor 20 minutes (4° C.; himac CF15, manufactured by Hitachi, Ltd.). Thesupernatant was recovered as a cell extract and the protein content wasdetermined using Protein Assay (manufactured by BioRad). The proteincontent of the cell extract made from the mouse bone marrow cells havingthe potential to differentiate into cardiomyocytes under the aboveconditions was found to be about 1 mg/ml.

[0445] The cell extract was then subjected to telomerase elongationreaction and PCR amplification according to the manufacture sinstructions. As the Taq polymerase, EX Taq polymerase (manufactured byTakara Shuzo) was used. After completion of the reactions, the sampleswere mixed with a {fraction (1/10)} volume of 10× staining solution(0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol) andsubjected to electrophoresis or 12.5% polyacrylamide gel (preparedaccording to the manufacture's instructions of TRAPeze TelomeraseDetection Kit) at a constant voltage of 250 mV. After theelectrophoresis, the gel was stained with Cyber Green (FMC) and analyzedusing a fluorescence image analyzer, FluoroImager (manufactured byMolecular Dynamics). The telomerase activity was detected in the samplesof the cell extracts having final concentration of 0.4-4 μg/ml.

EXAMPLE 15

[0446] Isolation and Culturing of Bone Marrow Cell having the Potentialto Differentiate into Cardiomyocyte from Rat Bone Marrow:

[0447] Six female Wistar rats of five week age (SLC Japan) weresubjected to cervical dislocation and then disinfected by sufficientlyapplying 70% ethanol. Next, the skin of each leg was incised in a broadrange and muscles covering the thighbone and shinbone were cut out toobtain the thighbone and shinbone. The thus obtained thighbone andshinbone were transferred into a culture dish of 10 cm in diameter(manufactured by Iwaki Glass) filled with PBS (manufactured by GibcoBRL) and muscles and joints were completely removed. Next, both ends ofthese bones were cut out using scissors, and the contents of bone marrowwere squeezed out with a water flow of a culture liquid (D-PBS,manufactured by Gibco BRL) using a 10 ml syringe (manufactured byTerumo) equipped with a 2OG needle. The thus obtained cell mass wasloosened into a homogeneous level by passing through the syringe. Thethus obtained cell suspension was recovered into a 50 ml capacitycentrifugation tube (manufactured by BECTON DICKINSON) and centrifugedat 1,500 rpm for 10 minutes (a low speed centrifuge manufactured byTOMY), and the precipitated cells were suspended in 6 ml of D-PBS. Whenthe number of cells was counted using a modified Neubauer countingchamber, the recovered cells were 2.6×10⁹ in total. This result meansthat 1×10⁸ cells were recovered from one thighbone or shinbone. The thusrecovered cells were diluted to a density of 1.3×10⁸ cells per 1 ml, 5ml of the resulting suspension was overlaid on a 1.073 g/ml Percoll(manufactured by Amersham Pharmacia Biotech)/D-PBS solution (25 ml)which had been put into a 50 ml capacity centrifugation tube, followedby centrifugation at room temperature and at 3,100 rpm for 30 minutes.After the centrifugation, cells were recovered from the interfacebetween the Percoll solution and cell suspension, diluted to 4 timeswith D-PBS and centrifuged at 2,300 rpm for 10 minutes and then the thusfractionated cells were recovered. The thus recovered cells weresuspended in IMDM medium (manufactured by Gibco BRL) containing 20% FCS,100 μg/ml penicillin, 250 ng/ml streptomycin and 85 μg/ml amphotericin(manufactured by Gibco BRL). When the number of cells at this stage wasagain counted, the recovered bone marrow cells were 4.7×10⁷ in total,meaning that cells corresponding to about 2% of the cells before thetreatment were recovered. The fractionated bone marrow cells were platedon three culture dishes for animal cells having a diameter of 10 cm(manufactured by Iwaki Glass, hereinafter referred to as “10-cm culturedish”) to a density of 2 to 5×105 cells/cm² and cultured at 33° C. in a5% CO₂-incubator (manufactured by Tabai). A half volume of the mediumwas exchanged with a fresh medium after 24 hours and 72 hours. Three or4 days thereafter, a half volume of the medium was exchanged with afresh medium. Since colonies became dense after a lapse of 15 days, thecells were removed with a trypsin EDTA treatment and a ⅔ part of themwas suspended in 4 ml of a stock solution (10% DMSO, 50% bone marrowcell culture supernatant and 40% the above medium which had not beenused), dispensed in 1 ml portions into 2 ml capacity tubes (manufacturedby Sumitomo Bakelite) and stored in a freezer, and the remaining ⅓ partwas again inoculated into two 10-cm culture dishes and subcultured.

EXAMPLE 16

[0448] Evaluation of Rat Bone Marrow-Derived Cell having the Potentialto Differentiate into Cardiomyocyte:

[0449] The rat bone marrow cells subcultured in the above were againremoved with the trypsin EDTA treatment when they became dense andinoculated into a 6 well plate (manufactured by BECTON DICKINSON) in5×10⁴ cells per well or into a 6 cm diameter culture dish coated withhuman fibronectin (Biocoat, manufactured by BECTON DICKINSON) in adensity of 1.3×10⁵ cells. One day thereafter, culturing was carried outunder two different conditions, one in which only 5-azacytidine(manufactured by Sigma, 10 μM in final concentration) was added, andanother in which 5-azacytidine, PDGF-BB (manufactured by Pepro Tech ECLTD, 10 ng/ml in final concentration) and all-trans retinoic acid (RA,manufactured by Sigma, 10⁻⁹ M in final concentration) were added, andthe medium was exchanged after 2 days of the culturing (in the latterconditions, PDGF and all-trans retinoic acid were again added at thetime of the medium exchange and after 2 days and 4 days). Three or 4days thereafter, the medium was exchanged, followed by culturing for 3weeks. As a result, differentiation of myotube-like cells was observedin the conditions in which 5-azacytidine, PDGF-BB and retinoic acid wereadded.

EXAMPLE 17

[0450] Forced Expression of Transcription Factor MesP1 and Enhancementof Cardiomyocyte Differentiation by Addition of Cytokine:

[0451] Influences of forced expression of a cardiomyocytedifferentiation-related transcription factor MesP1 in a bonemarrow-derived pluripotent stem cell (BMSC) having the potential todifferentiate into cardiomyocytes upon its differentiation intocardiomyocytes and influences of a combination of forced expression ofMesP1 with cytokine (FGF-8, ET-1, Midkine or BMP4) upon differentiationinto cardiomyocytes were examined.

[0452] A mouse bone marrow-derived pluripotent stem cell (BMSC-MesP1)having the potential to differentiate into cardiomyocytes in which theMesP1 gene was forced-expressed was obtained using a retrovirus vectorin the same manner as in Example 6, and then the differentiation wasinduced to examine efficiency of differentiation into cardiomyocytes.

[0453] The bone marrow cell (BMSC-MesP1) having the potential todifferentiate into cardiomyocytes in which MesP1 was forced expressedwas plated into a 60-mm culture dish in a density of 2×10⁴ cells/ml andcultured at 33° C. in a 5% CO₂-incubator. On the next day, 5-aza-C wasadded to the culture medium to give a final concentration of 3 μM,followed by culturing under five different conditions, namely (i)addition of FGF-8 to give a final concentration of 10 ng/ml (culturedish N), (ii) addition of ET-1 to give a final concentration of 10 ng/ml(culture dish P), (iii) addition of Midkine to give a finalconcentration of 10 ng/ml (culture dish Q), (iv) addition of BMP4 togive a final concentration of 10 ng/ml (culture dish R), and (v) noaddition (culture dish S).

[0454] On the next day, the medium was exchanged with a fresh medium inorder to eliminate 5-aza-C from the medium, and then the culturing wascontinued by adding FGF-8 to the culture dish N to give a finalconcentration of 10 ng/ml, ET-1 to the culture dish P to give a finalconcentration of 10 ng/ml, Midkine to the culture dish Q to give a finalconcentration of 10 ng/ml and BMP4 to the culture dish R to give a finalconcentration of 10 ng/ml. Two days and 4 days thereafter, the mediumexchange and addition of FGF-8, ET-1, Midkine or BMP4 were carried outsimilarly.

[0455] Four weeks after the addition of 5-aza-C, morphology of the cellswas observed under a phase contrast microscope. As a result, the numberof myotube-like cells was not changed greatly by the forced expressionof MesP1. In addition, about 50% of the cells became myotube-like cellsin the culture dish to which FGF-8, ET-1, Midkine or BMP4 had beenadded.

[0456] Next, RNA was recovered from the thus obtained myotube-likecells, and genes expressing in the myotube-like cells were analyzed byquantitative PCR using the synthetic oligonucleotides shown in SEQ IDNOS:71 to 78. As a result, expression of ANP as a gene specific for amyocardium was accelerated by the forced expression of MesP1. On theother hand, FGF-8, ET-1, Midkine or BMP4 did not further accelerate theexpression of ANP accelerated by the forced expression of MesP1.

[0457] Industrial Applicability

[0458] The present invention provides a bone marrow cell, a growthfactor, a vitamin and an adhesion molecule which are effective fortreating a heart disease accompanied with destruction and denaturationof a cardiomyocyte and for screening a therapeutic agent for it, andapplication methods thereof.

[0459] Free Text of Sequence Listings:

[0460] SEQ ID NO:33—Explanation of artificial sequence: Synthetic DNA

[0461] SEQ ID NO:34—Explanation of artificial sequence: Synthetic DNA

[0462] SEQ ID NO:35—Explanation of artificial sequence: Synthetic DNA

[0463] SEQ ID NO:36—Explanation of artificial sequence: Synthetic DNA

[0464] SEQ ID NO:37—Explanation of artificial sequence: Synthetic DNA

[0465] SEQ ID NO:38—Explanation of artificial sequence: Synthetic DNA

[0466] SEQ ID NO:39—Explanation of artificial sequence: Synthetic DNA

[0467] SEQ ID NO:40—Explanation of artificial sequence: Synthetic DNA

[0468] SEQ ID NO:41—Explanation of artificial sequence: Synthetic DNA

[0469] SEQ ID NO:42—Explanation of artificial sequence: Synthetic DNA

[0470] SEQ ID NO:43—Explanation of artificial sequence: Synthetic DNA

[0471] SEQ ID NO:44—Explanation of artificial sequence: Synthetic DNA

[0472] SEQ ID NO:45—Explanation of artificial sequence: Synthetic DNA

[0473] SEQ ID NO:46—Explanation of artificial sequence: Synthetic DNA

[0474] SEQ ID NO:47—Explanation of artificial sequence: Synthetic DNA

[0475] SEQ ID NO:48—Explanation of artificial sequence: Synthetic DNA

[0476] SEQ ID NO:49—Explanation of artificial sequence: Synthetic DNA

[0477] SEQ ID NO:50—Explanation of artificial sequence: Synthetic DNA

[0478] SEQ ID NO:51—Explanation of artificial sequence: Synthetic DNA

[0479] SEQ ID NO:52—Explanation of artificial sequence: Synthetic DNA

[0480] SEQ ID NO:53—Explanation of artificial sequence: Synthetic DNA

[0481] SEQ ID NO:54—Explanation of artificial sequence: Synthetic DNA

[0482] SEQ ID NO:55—Explanation of artificial sequence: Synthetic DNA

[0483] SEQ ID NO:56—Explanation of artificial sequence: Synthetic DNA

[0484] SEQ ID NO:57—Explanation of artificial sequence: Synthetic DNA

[0485] SEQ ID NO:58—Explanation of artificial sequence: Synthetic DNA

[0486] SEQ ID NO:59—Explanation of artificial sequence: Synthetic DNA

[0487] SEQ ID NO:60—Explanation of artificial sequence: Synthetic DNA

[0488] SEQ ID NO:61—Explanation of artificial sequence: Synthetic DNA

[0489] SEQ ID NO:62—Explanation of artificial sequence: Synthetic DNA

[0490] SEQ ID NO:63—Explanation of artificial sequence: Synthetic DNA

[0491] SEQ ID NO:64—Explanation of artificial sequence: Synthetic DNA

[0492] SEQ ID NO:65—Explanation of artificial sequence: Synthetic DNA

[0493] SEQ ID NO:66—Explanation of artificial sequence: Synthetic DNA

[0494] SEQ ID NO:67—Explanation of artificial sequence: Synthetic DNA

[0495] SEQ ID NO:68—Explanation of artificial sequence: Synthetic DNA

[0496] SEQ ID NO:69—Explanation of artificial sequence: Synthetic DNA

[0497] SEQ ID NO:70—Explanation of artificial sequence: Synthetic DNA

[0498] SEQ ID NO:71—Explanation of artificial sequence: Synthetic DNA

[0499] SEQ ID NO:72—Explanation of artificial sequence: Synthetic DNA

[0500] SEQ ID NO:73—Explanation of artificial sequence: Synthetic DNA

[0501] SEQ ID NO:74—Explanation of artificial sequence: Synthetic DNA

[0502] SEQ ID NO:75—Explanation of artificial sequence: Synthetic DNA

[0503] SEQ ID NO:76—Explanation of artificial sequence: Synthetic DNA

[0504] SEQ ID NO:77—Explanation of artificial sequence: Synthetic DNA

[0505] SEQ ID NO:78—Explanation of artificial sequence: Synthetic DNA

[0506] SEQ ID NO:79—Explanation of artificial sequence: Synthetic DNA

[0507] SEQ ID NO:80—Explanation of artificial sequence: Synthetic DNA

1 80 1 411 PRT Homo sapiens 1 Met Arg Ala His Pro Gly Gly Gly Arg CysCys Pro Glu Gln Glu Glu 1 5 10 15 Gly Glu Ser Ala Ala Gly Gly Ser GlyAla Gly Gly Asp Ser Ala Ile 20 25 30 Glu Gln Gly Gly Gln Gly Ser Ala LeuAla Pro Ser Pro Val Ser Gly 35 40 45 Val Arg Arg Glu Gly Ala Arg Gly GlyGly Arg Gly Arg Gly Arg Trp 50 55 60 Lys Gln Ala Gly Arg Gly Gly Gly ValCys Gly Arg Gly Arg Gly Arg 65 70 75 80 Gly Arg Gly Arg Gly Arg Gly ArgGly Arg Gly Arg Gly Arg Gly Arg 85 90 95 Pro Pro Ser Gly Gly Ser Gly LeuGly Gly Asp Gly Gly Gly Cys Gly 100 105 110 Gly Gly Gly Ser Gly Gly GlyGly Ala Pro Arg Arg Glu Pro Val Pro 115 120 125 Phe Pro Ser Gly Ser AlaGly Pro Gly Pro Arg Gly Pro Arg Ala Thr 130 135 140 Glu Ser Gly Lys ArgMet Asp Cys Pro Ala Leu Pro Pro Gly Trp Lys 145 150 155 160 Lys Glu GluVal Ile Arg Lys Ser Gly Leu Ser Ala Gly Lys Ser Asp 165 170 175 Val TyrTyr Phe Ser Pro Ser Gly Lys Lys Phe Arg Ser Lys Pro Gln 180 185 190 LeuAla Arg Tyr Leu Gly Asn Thr Val Asp Leu Ser Ser Phe Asp Phe 195 200 205Arg Thr Gly Lys Met Met Pro Ser Lys Leu Gln Lys Asn Lys Gln Arg 210 215220 Leu Arg Asn Asp Pro Leu Asn Gln Asn Lys Gly Lys Pro Asp Leu Asn 225230 235 240 Thr Thr Leu Pro Ile Arg Gln Thr Ala Ser Ile Phe Lys Gln ProVal 245 250 255 Thr Lys Val Thr Asn His Pro Ser Asn Lys Val Lys Ser AspPro Gln 260 265 270 Arg Met Asn Glu Gln Pro Arg Gln Leu Phe Trp Glu LysArg Leu Gln 275 280 285 Gly Leu Ser Ala Ser Asp Val Thr Glu Gln Ile IleLys Thr Met Glu 290 295 300 Leu Pro Lys Gly Leu Gln Gly Val Gly Pro GlySer Asn Asp Glu Thr 305 310 315 320 Leu Leu Ser Ala Val Ala Ser Ala LeuHis Thr Ser Ser Ala Pro Ile 325 330 335 Thr Gly Gln Val Ser Ala Ala ValGlu Lys Asn Pro Ala Val Trp Leu 340 345 350 Asn Thr Ser Gln Pro Leu CysLys Ala Phe Ile Val Thr Asp Glu Asp 355 360 365 Ile Arg Lys Gln Glu GluArg Val Gln Gln Val Arg Lys Lys Leu Glu 370 375 380 Glu Ala Leu Met AlaAsp Ile Leu Ser Arg Ala Ala Asp Thr Glu Glu 385 390 395 400 Met Asp IleGlu Met Asp Ser Gly Asp Glu Ala 405 410 2 1233 DNA Homo sapiens CDS(1)..(1236) 2 atg cgc gcg cac ccg ggg gga ggc cgc tgc tgc ccg gag caggag gag 48 Met Arg Ala His Pro Gly Gly Gly Arg Cys Cys Pro Glu Gln GluGlu 1 5 10 15 ggg gag agt gcg gcg ggc ggc agc ggc gct ggc ggc gac tccgcc ata 96 Gly Glu Ser Ala Ala Gly Gly Ser Gly Ala Gly Gly Asp Ser AlaIle 20 25 30 gag cag ggg ggc cag ggc agc gcg ctc gcc ccg tcc ccg gtg agcggc 144 Glu Gln Gly Gly Gln Gly Ser Ala Leu Ala Pro Ser Pro Val Ser Gly35 40 45 gtg cgc agg gaa ggc gct cgg ggc ggc ggc cgt ggc cgg ggg cgg tgg192 Val Arg Arg Glu Gly Ala Arg Gly Gly Gly Arg Gly Arg Gly Arg Trp 5055 60 aag cag gcg ggc cgg ggc ggc ggc gtc tgt ggc cgt ggc cgg ggc cgg240 Lys Gln Ala Gly Arg Gly Gly Gly Val Cys Gly Arg Gly Arg Gly Arg 6570 75 80 ggc cgt ggc cgg gga cgg gga cgg ggc cgg ggc cgg ggc cgc ggc cgt288 Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg 8590 95 ccc ccg agt ggc ggc agc ggc ctt ggc ggc gac ggc ggc ggc tgc ggc336 Pro Pro Ser Gly Gly Ser Gly Leu Gly Gly Asp Gly Gly Gly Cys Gly 100105 110 ggc ggc ggc agc ggt ggc ggc ggc gcc ccc cgg cgg gag ccg gtc cct384 Gly Gly Gly Ser Gly Gly Gly Gly Ala Pro Arg Arg Glu Pro Val Pro 115120 125 ttc ccg tcg ggg agc gcg ggg ccg ggg ccc agg gga ccc cgg gcc acg432 Phe Pro Ser Gly Ser Ala Gly Pro Gly Pro Arg Gly Pro Arg Ala Thr 130135 140 gag agc ggg aag agg atg gat tgc ccg gcc ctc ccc ccc gga tgg aag480 Glu Ser Gly Lys Arg Met Asp Cys Pro Ala Leu Pro Pro Gly Trp Lys 145150 155 160 aag gag gaa gtg atc cga aaa tct ggg cta agt gct ggc aag agcgat 528 Lys Glu Glu Val Ile Arg Lys Ser Gly Leu Ser Ala Gly Lys Ser Asp165 170 175 gtc tac tac ttc agt cca agt ggt aag aag ttc aga agc aag cctcag 576 Val Tyr Tyr Phe Ser Pro Ser Gly Lys Lys Phe Arg Ser Lys Pro Gln180 185 190 ttg gca agg tac ctg gga aat act gtt gat ctc agc agt ttt gacttc 624 Leu Ala Arg Tyr Leu Gly Asn Thr Val Asp Leu Ser Ser Phe Asp Phe195 200 205 aga act gga aag atg atg cct agt aaa tta cag aag aac aaa cagaga 672 Arg Thr Gly Lys Met Met Pro Ser Lys Leu Gln Lys Asn Lys Gln Arg210 215 220 ctg cga aac gat cct ctc aat caa aat aag ggt aaa cca gac ttgaat 720 Leu Arg Asn Asp Pro Leu Asn Gln Asn Lys Gly Lys Pro Asp Leu Asn225 230 235 240 aca aca ttg cca att aga caa aca gca tca att ttc aaa caaccg gta 768 Thr Thr Leu Pro Ile Arg Gln Thr Ala Ser Ile Phe Lys Gln ProVal 245 250 255 acc aaa gtc aca aat cat cct agt aat aaa gtg aaa tca gaccca caa 816 Thr Lys Val Thr Asn His Pro Ser Asn Lys Val Lys Ser Asp ProGln 260 265 270 cga atg aat gaa cag cca cgt cag ctt ttc tgg gag aag aggcta caa 864 Arg Met Asn Glu Gln Pro Arg Gln Leu Phe Trp Glu Lys Arg LeuGln 275 280 285 gga ctt agt gca tca gat gta aca gaa caa att ata aaa accatg gaa 912 Gly Leu Ser Ala Ser Asp Val Thr Glu Gln Ile Ile Lys Thr MetGlu 290 295 300 cta ccc aaa ggt ctt caa gga gtt ggt cca ggt agc aat gatgag acc 960 Leu Pro Lys Gly Leu Gln Gly Val Gly Pro Gly Ser Asn Asp GluThr 305 310 315 320 ctt tta tct gct gtt gcc agt gct ttg cac aca agc tctgcg cca atc 1008 Leu Leu Ser Ala Val Ala Ser Ala Leu His Thr Ser Ser AlaPro Ile 325 330 335 aca ggg caa gtc tcc gct gct gtg gaa aag aac cct gctgtt tgg ctt 1056 Thr Gly Gln Val Ser Ala Ala Val Glu Lys Asn Pro Ala ValTrp Leu 340 345 350 aac aca tct caa ccc ctc tgc aaa gct ttt att gtc acagat gaa gac 1104 Asn Thr Ser Gln Pro Leu Cys Lys Ala Phe Ile Val Thr AspGlu Asp 355 360 365 atc agg aaa cag gaa gag cga gta cag caa gta cgc aagaaa ttg gaa 1152 Ile Arg Lys Gln Glu Glu Arg Val Gln Gln Val Arg Lys LysLeu Glu 370 375 380 gaa gca ctg atg gca gac atc ttg tcg cga gct gct gataca gaa gag 1200 Glu Ala Leu Met Ala Asp Ile Leu Ser Arg Ala Ala Asp ThrGlu Glu 385 390 395 400 atg gat att gaa atg gac agt gga gat gaa gcc 1233Met Asp Ile Glu Met Asp Ser Gly Asp Glu Ala 405 410 3 196 PRT Homosapiens 3 Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr LeuAla 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val IleGlu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln ArgLeu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr SerLeu Arg 50 55 60 Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys ArgPro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val ProAla Val Cys 85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser GlnVal Asp Pro 100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys ValGlu Val Lys Arg 115 120 125 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val LysCys Gln Pro Ser Arg 130 135 140 Val His His Arg Ser Val Lys Val Ala LysVal Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val GlnVal Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys Ala Thr Thr SerLeu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Asp Val Arg 195 4588 DNA Homo sapiens CDS (1)..(591) 4 atg agg acc ttg gct tgc ctg ctgctc ctc ggc tgc gga tac ctc gcc 48 Met Arg Thr Leu Ala Cys Leu Leu LeuLeu Gly Cys Gly Tyr Leu Ala 1 5 10 15 cat gtt ctg gcc gag gaa gcc gagatc ccc cgc gag gtg atc gag agg 96 His Val Leu Ala Glu Glu Ala Glu IlePro Arg Glu Val Ile Glu Arg 20 25 30 ctg gcc cgc agt cag atc cac agc atccgg gac ctc cag cga ctc ctg 144 Leu Ala Arg Ser Gln Ile His Ser Ile ArgAsp Leu Gln Arg Leu Leu 35 40 45 gag ata gac tcc gta ggg agt gag gat tctttg gac acc agc ctg aga 192 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser LeuAsp Thr Ser Leu Arg 50 55 60 gct cac ggg gtc cac gcc act aag cat gtg cccgag aag cgg ccc ctg 240 Ala His Gly Val His Ala Thr Lys His Val Pro GluLys Arg Pro Leu 65 70 75 80 ccc att cgg agg aag aga agc atc gag gaa gctgtc ccc gct gtc tgc 288 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala ValPro Ala Val Cys 85 90 95 aag acc agg acg gtc att tac gag att cct cgg agtcag gtc gac ccc 336 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser GlnVal Asp Pro 100 105 110 acg tcc gcc aac ttc ctg atc tgg ccc ccg tgc gtggag gtg aaa cgc 384 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val GluVal Lys Arg 115 120 125 tgc acc ggc tgc tgc aac acg agc agt gtc aag tgccag ccc tcc cgc 432 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys GlnPro Ser Arg 130 135 140 gtc cac cac cgc agc gtc aag gtg gcc aag gtg gaatac gtc agg aag 480 Val His His Arg Ser Val Lys Val Ala Lys Val Glu TyrVal Arg Lys 145 150 155 160 aag cca aaa tta aaa gaa gtc cag gtg agg ttagag gag cat ttg gag 528 Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu GluGlu His Leu Glu 165 170 175 tgc gcc tgc gcg acc aca agc ctg aat ccg gattat cgg gaa gag gac 576 Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp TyrArg Glu Glu Asp 180 185 190 acg gat gtg agg 588 Thr Asp Val Arg 195 5241 PRT Homo sapiens 5 Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu CysCys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro GluGlu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp AspLeu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala GluLeu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu SerLeu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala GluPro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu IleSer Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val TrpPro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys Asn AsnArg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro ValGln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile PheLys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys CysGlu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly GlySer Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr IleArg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 LysPhe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235240 Ala 6 723 DNA Homo sapiens CDS (1)..(726) 6 atg aat cgc tgc tgg gcgctc ttc ctg tct ctc tgc tgc tac ctg cgt 48 Met Asn Arg Cys Trp Ala LeuPhe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 ctg gtc agc gcc gag ggggac ccc att ccc gag gag ctt tat gag atg 96 Leu Val Ser Ala Glu Gly AspPro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 ctg agt gac cac tcg atc cgctcc ttt gat gat ctc caa cgc ctg ctg 144 Leu Ser Asp His Ser Ile Arg SerPhe Asp Asp Leu Gln Arg Leu Leu 35 40 45 cac gga gac ccc gga gag gaa gatggg gcc gag ttg gac ctg aac atg 192 His Gly Asp Pro Gly Glu Glu Asp GlyAla Glu Leu Asp Leu Asn Met 50 55 60 acc cgc tcc cac tct gga ggc gag ctggag agc ttg gct cgt gga aga 240 Thr Arg Ser His Ser Gly Gly Glu Leu GluSer Leu Ala Arg Gly Arg 65 70 75 80 agg agc ctg ggt tcc ctg acc att gctgag ccg gcc atg atc gcc gag 288 Arg Ser Leu Gly Ser Leu Thr Ile Ala GluPro Ala Met Ile Ala Glu 85 90 95 tgc aag acg cgc acc gag gtg ttc gag atctcc cgg cgc ctc ata gac 336 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile SerArg Arg Leu Ile Asp 100 105 110 cgc acc aac gcc aac ttc ctg gtg tgg ccgccc tgt gtg gag gtg cag 384 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro ProCys Val Glu Val Gln 115 120 125 cgc tgc tcc ggc tgc tgc aac aac cgc aacgtg cag tgc cgc ccc acc 432 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn ValGln Cys Arg Pro Thr 130 135 140 cag gtg cag ctg cga cct gtc cag gtg agaaag atc gag att gtg cgg 480 Gln Val Gln Leu Arg Pro Val Gln Val Arg LysIle Glu Ile Val Arg 145 150 155 160 aag aag cca atc ttt aag aag gcc acggtg acg ctg gaa gac cac ctg 528 Lys Lys Pro Ile Phe Lys Lys Ala Thr ValThr Leu Glu Asp His Leu 165 170 175 gca tgc aag tgt gag aca gtg gca gctgca cgg cct gtg acc cga agc 576 Ala Cys Lys Cys Glu Thr Val Ala Ala AlaArg Pro Val Thr Arg Ser 180 185 190 ccg ggg ggt tcc cag gag cag cga gccaaa acg ccc caa act cgg gtg 624 Pro Gly Gly Ser Gln Glu Gln Arg Ala LysThr Pro Gln Thr Arg Val 195 200 205 acc att cgg acg gtg cga gtc cgc cggccc ccc aag ggc aag cac cgg 672 Thr Ile Arg Thr Val Arg Val Arg Arg ProPro Lys Gly Lys His Arg 210 215 220 aaa ttc aag cac acg cat gac aag acggca ctg aag gag acc ctt gga 720 Lys Phe Lys His Thr His Asp Lys Thr AlaLeu Lys Glu Thr Leu Gly 225 230 235 240 gcc 723 Ala 7 155 PRT Homosapiens 7 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu AspGly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro LysArg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro AspGly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys LeuGln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val CysAla Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu AlaSer Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu SerAsn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp TyrVal Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys ThrGly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala LysSer 145 150 155 8 465 DNA Homo sapiens CDS (1)..(468) 8 atg gca gcc gggagc atc acc acg ctg ccc gcc ttg ccc gag gat ggc 48 Met Ala Ala Gly SerIle Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 ggc agc ggc gccttc ccg ccc ggc cac ttc aag gac ccc aag cgg ctg 96 Gly Ser Gly Ala PhePro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 tac tgc aaa aac gggggc ttc ttc ctg cgc atc cac ccc gac ggc cga 144 Tyr Cys Lys Asn Gly GlyPhe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 gtt gac ggg gtc cgg gagaag agc gac cct cac atc aag cta caa ctt 192 Val Asp Gly Val Arg Glu LysSer Asp Pro His Ile Lys Leu Gln Leu 50 55 60 caa gca gaa gag aga gga gttgtg tct atc aaa gga gtg tgt gct aac 240 Gln Ala Glu Glu Arg Gly Val ValSer Ile Lys Gly Val Cys Ala Asn 65 70 75 80 cgt tac ctg gct atg aag gaagat gga aga tta ctg gct tct aaa tgt 288 Arg Tyr Leu Ala Met Lys Glu AspGly Arg Leu Leu Ala Ser Lys Cys 85 90 95 gtt acg gat gag tgt ttc ttt tttgaa cga ttg gaa tct aat aac tac 336 Val Thr Asp Glu Cys Phe Phe Phe GluArg Leu Glu Ser Asn Asn Tyr 100 105 110 aat act tac cgg tca agg aaa tacacc agt tgg tat gtg gca ttg aaa 384 Asn Thr Tyr Arg Ser Arg Lys Tyr ThrSer Trp Tyr Val Ala Leu Lys 115 120 125 cga act ggg cag tat aaa ctt ggatcc aaa aca gga cct ggg cag aaa 432 Arg Thr Gly Gln Tyr Lys Leu Gly SerLys Thr Gly Pro Gly Gln Lys 130 135 140 gct ata ctt ttt ctt cca atg tctgct aag agc 465 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 1559 324 PRT Homo sapiens 9 Met Phe Pro Ser Pro Ala Leu Thr Pro Thr Pro PheSer Val Lys Asp 1 5 10 15 Ile Leu Asn Leu Glu Gln Gln Gln Arg Ser LeuAla Ala Ala Gly Glu 20 25 30 Leu Ser Ala Arg Leu Glu Ala Thr Leu Ala ProSer Ser Cys Met Leu 35 40 45 Ala Ala Phe Lys Pro Glu Ala Tyr Ala Gly ProGlu Ala Ala Ala Pro 50 55 60 Gly Leu Pro Glu Leu Arg Ala Glu Leu Gly ArgAla Pro Ser Pro Ala 65 70 75 80 Lys Cys Ala Ser Ala Phe Pro Ala Ala ProAla Phe Tyr Pro Arg Ala 85 90 95 Tyr Ser Asp Pro Asp Pro Ala Lys Asp ProArg Ala Glu Lys Lys Glu 100 105 110 Leu Cys Ala Leu Gln Lys Ala Val GluLeu Glu Lys Thr Glu Ala Asp 115 120 125 Asn Ala Glu Arg Pro Arg Ala ArgArg Arg Arg Lys Pro Arg Val Leu 130 135 140 Phe Ser Gln Ala Gln Val TyrGlu Leu Glu Arg Arg Phe Lys Gln Gln 145 150 155 160 Arg Tyr Leu Ser AlaPro Glu Arg Asp Gln Leu Ala Ser Val Leu Lys 165 170 175 Leu Thr Ser ThrGln Val Lys Ile Trp Phe Gln Asn Arg Arg Tyr Lys 180 185 190 Cys Lys ArgGln Arg Gln Asp Gln Thr Leu Glu Leu Val Gly Leu Pro 195 200 205 Pro ProPro Pro Pro Pro Ala Arg Arg Ile Ala Val Pro Val Leu Val 210 215 220 ArgAsp Gly Lys Pro Cys Leu Gly Asp Ser Ala Pro Tyr Ala Pro Ala 225 230 235240 Tyr Gly Val Gly Leu Asn Pro Tyr Gly Tyr Asn Ala Tyr Pro Ala Tyr 245250 255 Pro Gly Tyr Gly Gly Ala Ala Cys Ser Pro Gly Tyr Ser Cys Thr Ala260 265 270 Ala Tyr Pro Ala Gly Pro Ser Pro Ala Gln Pro Ala Thr Ala AlaAla 275 280 285 Asn Asn Asn Phe Val Asn Phe Gly Val Gly Asp Leu Asn AlaVal Gln 290 295 300 Ser Pro Gly Ile Pro Gln Ser Asn Ser Gly Val Ser ThrLeu His Gly 305 310 315 320 Ile Arg Ala Trp 10 972 DNA Homo sapiens CDS(1)..(975) 10 atg ttc ccc agc cct gct ctc acg ccc acg ccc ttc tca gtcaaa gac 48 Met Phe Pro Ser Pro Ala Leu Thr Pro Thr Pro Phe Ser Val LysAsp 1 5 10 15 atc cta aac ctg gaa cag cag cag cgc agc ctg gct gcc gccgga gag 96 Ile Leu Asn Leu Glu Gln Gln Gln Arg Ser Leu Ala Ala Ala GlyGlu 20 25 30 ctc tct gcc cgc ctg gag gcg acc ctg gcg ccc tcc tcc tgc atgctg 144 Leu Ser Ala Arg Leu Glu Ala Thr Leu Ala Pro Ser Ser Cys Met Leu35 40 45 gcc gcc ttc aag cca gag gcc tac gct ggg ccc gag gcg gct gcg ccg192 Ala Ala Phe Lys Pro Glu Ala Tyr Ala Gly Pro Glu Ala Ala Ala Pro 5055 60 ggc ctc cca gag ctg cgc gca gag ctg ggc cgc gcg cct tca ccg gcc240 Gly Leu Pro Glu Leu Arg Ala Glu Leu Gly Arg Ala Pro Ser Pro Ala 6570 75 80 aag tgt gcg tct gcc ttt ccc gcc gcc ccc gcc ttc tat cca cgt gcc288 Lys Cys Ala Ser Ala Phe Pro Ala Ala Pro Ala Phe Tyr Pro Arg Ala 8590 95 tac agc gac ccc gac cca gcc aag gac cct aga gcc gaa aag aaa gag336 Tyr Ser Asp Pro Asp Pro Ala Lys Asp Pro Arg Ala Glu Lys Lys Glu 100105 110 ctg tgc gcg ctg cag aag gcg gtg gag ctg gag aag aca gag gcg gac384 Leu Cys Ala Leu Gln Lys Ala Val Glu Leu Glu Lys Thr Glu Ala Asp 115120 125 aac gcg gag cgg ccc cgg gcg cga cgg cgg agg aag ccg cgc gtg ctc432 Asn Ala Glu Arg Pro Arg Ala Arg Arg Arg Arg Lys Pro Arg Val Leu 130135 140 ttc tcg cag gcg cag gtc tat gag ctg gag cgg cgc ttc aag cag cag480 Phe Ser Gln Ala Gln Val Tyr Glu Leu Glu Arg Arg Phe Lys Gln Gln 145150 155 160 cgg tac ctg tcg gcc ccc gaa cgc gac cag ctg gcc agc gtg ctgaaa 528 Arg Tyr Leu Ser Ala Pro Glu Arg Asp Gln Leu Ala Ser Val Leu Lys165 170 175 ctc acg tcc acg cag gtc aag atc tgg ttc cag aac cgg cgc tacaag 576 Leu Thr Ser Thr Gln Val Lys Ile Trp Phe Gln Asn Arg Arg Tyr Lys180 185 190 tgc aag cgg cag cgg cag gac cag act ctg gag ctg gtg ggg ctgccc 624 Cys Lys Arg Gln Arg Gln Asp Gln Thr Leu Glu Leu Val Gly Leu Pro195 200 205 ccg ccg ccg ccg ccg cct gcc cgc agg atc gcg gtg cca gtg ctggtg 672 Pro Pro Pro Pro Pro Pro Ala Arg Arg Ile Ala Val Pro Val Leu Val210 215 220 cgc gat ggc aag cca tgc cta ggg gac tcg gcg ccc tac gcg cctgcc 720 Arg Asp Gly Lys Pro Cys Leu Gly Asp Ser Ala Pro Tyr Ala Pro Ala225 230 235 240 tac ggc gtg ggc ctc aat ccc tac ggt tat aac gcc tac cccgcc tat 768 Tyr Gly Val Gly Leu Asn Pro Tyr Gly Tyr Asn Ala Tyr Pro AlaTyr 245 250 255 ccg ggt tac ggc ggc gcg gcc tgc agc cct ggc tac agc tgcact gcc 816 Pro Gly Tyr Gly Gly Ala Ala Cys Ser Pro Gly Tyr Ser Cys ThrAla 260 265 270 gct tac ccc gcc ggg cct tcc cca gcg cag ccg gcc act gccgcc gcc 864 Ala Tyr Pro Ala Gly Pro Ser Pro Ala Gln Pro Ala Thr Ala AlaAla 275 280 285 aac aac aac ttc gtg aac ttc ggc gtc ggg gac ttg aat gcggtt cag 912 Asn Asn Asn Phe Val Asn Phe Gly Val Gly Asp Leu Asn Ala ValGln 290 295 300 agc ccc ggg att ccg cag agc aac tcg gga gtg tcc acg ctgcat ggt 960 Ser Pro Gly Ile Pro Gln Ser Asn Ser Gly Val Ser Thr Leu HisGly 305 310 315 320 atc cga gcc tgg 972 Ile Arg Ala Trp 324 11 442 PRTHomo sapiens 11 Met Tyr Gln Ser Leu Ala Met Ala Ala Asn His Gly Pro ProPro Gly 1 5 10 15 Ala Tyr Gln Ala Gly Gly Pro Gly Pro Phe Met His GlyAla Gly Ala 20 25 30 Ala Ser Ser Pro Val Tyr Leu Pro Thr Pro Arg Val ProSer Ser Val 35 40 45 Leu Gly Leu Ser Tyr Leu Gln Gly Gly Gly Ala Gly SerAla Ser Gly 50 55 60 Gly Pro Ser Gly Gly Ser Pro Gly Gly Ala Ala Ser GlyAla Gly Pro 65 70 75 80 Gly Thr Gln Gln Gly Ser Pro Gly Trp Ser Gln AlaGly Ala Thr Gly 85 90 95 Ala Ala Tyr Thr Pro Pro Pro Val Ser Pro Arg PheSer Phe Pro Gly 100 105 110 Thr Thr Gly Ser Leu Ala Ala Ala Ala Ala AlaAla Ala Ala Arg Glu 115 120 125 Ala Ala Ala Tyr Ser Ser Gly Gly Gly AlaAla Gly Ala Gly Leu Ala 130 135 140 Gly Arg Glu Gln Tyr Gly Arg Ala GlyPhe Ala Gly Ser Tyr Ser Ser 145 150 155 160 Pro Tyr Pro Ala Tyr Met AlaAsp Val Gly Ala Ser Trp Ala Ala Ala 165 170 175 Ala Ala Ala Ser Ala GlyPro Phe Asp Ser Pro Val Leu His Ser Leu 180 185 190 Pro Gly Arg Ala AsnPro Ala Ala Arg His Pro Asn Leu Asp Met Phe 195 200 205 Asp Asp Phe SerGlu Gly Arg Glu Cys Val Asn Cys Gly Ala Met Ser 210 215 220 Thr Pro LeuTrp Arg Arg Asp Gly Thr Gly His Tyr Leu Cys Asn Ala 225 230 235 240 CysGly Leu Tyr His Lys Met Asn Gly Ile Asn Arg Pro Leu Ile Lys 245 250 255Pro Gln Arg Arg Leu Ser Ala Ser Arg Arg Val Gly Leu Ser Cys Ala 260 265270 Asn Cys Gln Thr Thr Thr Thr Thr Leu Trp Arg Arg Asn Ala Glu Gly 275280 285 Glu Pro Val Cys Asn Ala Cys Gly Leu Tyr Met Lys Leu His Gly Val290 295 300 Pro Arg Pro Leu Ala Met Arg Lys Glu Gly Ile Gln Thr Arg LysArg 305 310 315 320 Lys Pro Lys Asn Leu Asn Lys Ser Lys Thr Pro Ala AlaPro Ser Gly 325 330 335 Ser Glu Ser Leu Pro Pro Ala Ser Gly Ala Ser SerAsn Ser Ser Asn 340 345 350 Ala Thr Thr Ser Ser Ser Glu Glu Met Arg ProIle Lys Thr Glu Pro 355 360 365 Gly Leu Ser Ser His Tyr Gly His Ser SerSer Val Ser Gln Thr Phe 370 375 380 Ser Val Ser Ala Met Ser Gly His GlyPro Ser Ile His Pro Val Leu 385 390 395 400 Ser Ala Leu Lys Leu Ser ProGln Gly Tyr Ala Ser Pro Val Ser Gln 405 410 415 Ser Pro Gln Thr Ser SerLys Gln Asp Ser Trp Asn Ser Leu Val Leu 420 425 430 Ala Asp Ser His GlyAsp Ile Ile Thr Ala 435 440 12 1326 DNA Homo sapiens CDS (1)..(1329) 12atg tat cag agc ttg gcc atg gcc gcc aac cac ggg ccg ccc ccc ggt 48 MetTyr Gln Ser Leu Ala Met Ala Ala Asn His Gly Pro Pro Pro Gly 1 5 10 15gcc tac cag gcg ggc ggc ccc ggc ccc ttc atg cac ggc gcg ggc gcc 96 AlaTyr Gln Ala Gly Gly Pro Gly Pro Phe Met His Gly Ala Gly Ala 20 25 30 gcgtcc tcg cca gtc tac ctg ccc aca ccg cgg gtg ccc tcc tcc gtt 144 Ala SerSer Pro Val Tyr Leu Pro Thr Pro Arg Val Pro Ser Ser Val 35 40 45 ctg ggcctg tcc tac ctc cag ggc gga ggc gcg ggc tct gcg tcc gga 192 Leu Gly LeuSer Tyr Leu Gln Gly Gly Gly Ala Gly Ser Ala Ser Gly 50 55 60 ggc ccc tcgggc ggc agc ccc ggt ggg gcc gcg tct ggt gcg ggg ccc 240 Gly Pro Ser GlyGly Ser Pro Gly Gly Ala Ala Ser Gly Ala Gly Pro 65 70 75 80 ggg acc cagcag ggc agc ccg gga tgg agc cag gcg gga gcg acc gga 288 Gly Thr Gln GlnGly Ser Pro Gly Trp Ser Gln Ala Gly Ala Thr Gly 85 90 95 gcc gct tac accccg ccg ccg gtg tcg ccg cgc ttc tcc ttc ccg ggg 336 Ala Ala Tyr Thr ProPro Pro Val Ser Pro Arg Phe Ser Phe Pro Gly 100 105 110 acc acc ggg tccctg gcg gcg gcg gcg gcg gct gcc gcc gcc cgg gaa 384 Thr Thr Gly Ser LeuAla Ala Ala Ala Ala Ala Ala Ala Ala Arg Glu 115 120 125 gct gcg gcc tacagc agt ggc ggc gga gcg gcg ggt gcg ggc ctg gcg 432 Ala Ala Ala Tyr SerSer Gly Gly Gly Ala Ala Gly Ala Gly Leu Ala 130 135 140 ggc cgc gag cagtac ggg cgc gcc ggc ttc gcg ggc tcc tac tcc agc 480 Gly Arg Glu Gln TyrGly Arg Ala Gly Phe Ala Gly Ser Tyr Ser Ser 145 150 155 160 ccc tac ccggct tac atg gcc gac gtg ggc gcg tcc tgg gcc gca gcc 528 Pro Tyr Pro AlaTyr Met Ala Asp Val Gly Ala Ser Trp Ala Ala Ala 165 170 175 gcc gcc gcctcc gcc ggc ccc ttc gac agc ccg gtc ctg cac agc ctg 576 Ala Ala Ala SerAla Gly Pro Phe Asp Ser Pro Val Leu His Ser Leu 180 185 190 ccc ggc cgggcc aac ccg gcc gcc cga cac ccc aat ctc gat atg ttt 624 Pro Gly Arg AlaAsn Pro Ala Ala Arg His Pro Asn Leu Asp Met Phe 195 200 205 gac gac ttctca gaa ggc aga gag tgt gtc aac tgt ggg gct atg tcc 672 Asp Asp Phe SerGlu Gly Arg Glu Cys Val Asn Cys Gly Ala Met Ser 210 215 220 acc ccg ctctgg agg cga gat ggg acg ggt cac tat ctg tgc aac gcc 720 Thr Pro Leu TrpArg Arg Asp Gly Thr Gly His Tyr Leu Cys Asn Ala 225 230 235 240 tgt ggcctc tac cac aag atg aac ggc atc aac cgg ccg ctc atc aag 768 Cys Gly LeuTyr His Lys Met Asn Gly Ile Asn Arg Pro Leu Ile Lys 245 250 255 cct cagcgc cgg ctg tcc gcc tcc cgc cga gtg ggc ctc tcc tgt gcc 816 Pro Gln ArgArg Leu Ser Ala Ser Arg Arg Val Gly Leu Ser Cys Ala 260 265 270 aac tgccag acc acc acc acc acg ctg tgg cgc cgc aat gcg gag ggc 864 Asn Cys GlnThr Thr Thr Thr Thr Leu Trp Arg Arg Asn Ala Glu Gly 275 280 285 gag cctgtg tgc aat gcc tgc ggc ctc tac atg aag ctc cac ggg gtg 912 Glu Pro ValCys Asn Ala Cys Gly Leu Tyr Met Lys Leu His Gly Val 290 295 300 ccc aggcct ctt gca atg cgg aaa gag ggg atc caa acc aga aaa cgg 960 Pro Arg ProLeu Ala Met Arg Lys Glu Gly Ile Gln Thr Arg Lys Arg 305 310 315 320 aagccc aag aac ctg aat aaa tct aag aca cca gca gct cct tca ggc 1008 Lys ProLys Asn Leu Asn Lys Ser Lys Thr Pro Ala Ala Pro Ser Gly 325 330 335 agtgag agc ctt cct ccc gcc agc ggt gct tcc agc aac tcc agc aac 1056 Ser GluSer Leu Pro Pro Ala Ser Gly Ala Ser Ser Asn Ser Ser Asn 340 345 350 gccacc acc agc agc agc gag gag atg cgt ccc atc aag acg gag cct 1104 Ala ThrThr Ser Ser Ser Glu Glu Met Arg Pro Ile Lys Thr Glu Pro 355 360 365 ggcctg tca tct cac tac ggg cac agc agc tcc gtg tcc cag acg ttc 1152 Gly LeuSer Ser His Tyr Gly His Ser Ser Ser Val Ser Gln Thr Phe 370 375 380 tcagtc agt gcg atg tct ggc cat ggg ccc tcc atc cac cct gtc ctc 1200 Ser ValSer Ala Met Ser Gly His Gly Pro Ser Ile His Pro Val Leu 385 390 395 400tcg gcc ctg aag ctc tcc cca caa ggc tat gcg tct ccc gtc agc cag 1248 SerAla Leu Lys Leu Ser Pro Gln Gly Tyr Ala Ser Pro Val Ser Gln 405 410 415tct cca cag acc agc tcc aag cag gac tct tgg aac agt ctg gtc ttg 1296 SerPro Gln Thr Ser Ser Lys Gln Asp Ser Trp Asn Ser Leu Val Leu 420 425 430gcc gac agt cac ggg gac ata atc act gcg 1326 Ala Asp Ser His Gly Asp IleIle Thr Ala 435 440 13 507 PRT Homo sapiens 13 Met Gly Arg Lys Lys IleGln Ile Thr Arg Ile Met Asp Glu Arg Asn 1 5 10 15 Arg Gln Val Thr PheThr Lys Arg Lys Phe Gly Leu Met Lys Lys Ala 20 25 30 Tyr Glu Leu Ser ValLeu Cys Asp Cys Glu Ile Ala Leu Ile Ile Phe 35 40 45 Asn Ser Ser Asn LysLeu Phe Gln Tyr Ala Ser Thr Asp Met Asp Lys 50 55 60 Val Leu Leu Lys TyrThr Glu Tyr Asn Glu Pro His Glu Ser Arg Thr 65 70 75 80 Asn Ser Asp IleVal Glu Ala Leu Asn Lys Lys Glu His Arg Gly Cys 85 90 95 Asp Ser Pro AspPro Asp Thr Ser Tyr Val Leu Thr Pro His Thr Glu 100 105 110 Glu Lys TyrLys Lys Ile Asn Glu Glu Phe Asp Asn Met Met Arg Asn 115 120 125 His LysIle Ala Pro Gly Leu Pro Pro Gln Asn Phe Ser Met Ser Val 130 135 140 ThrVal Pro Val Thr Ser Pro Asn Ala Leu Ser Tyr Thr Asn Pro Gly 145 150 155160 Ser Ser Leu Val Ser Pro Ser Leu Ala Ala Ser Ser Thr Leu Thr Asp 165170 175 Ser Ser Met Leu Ser Pro Pro Gln Thr Thr Leu His Arg Asn Val Ser180 185 190 Pro Gly Ala Pro Gln Arg Pro Pro Ser Thr Gly Asn Ala Gly GlyMet 195 200 205 Leu Ser Thr Thr Asp Leu Thr Val Pro Asn Gly Ala Gly SerSer Pro 210 215 220 Val Gly Asn Gly Phe Val Asn Ser Arg Ala Ser Pro AsnLeu Ile Gly 225 230 235 240 Ala Thr Gly Ala Asn Ser Leu Gly Lys Val MetPro Thr Lys Ser Pro 245 250 255 Pro Pro Pro Gly Gly Gly Asn Leu Gly MetAsn Ser Arg Lys Pro Asp 260 265 270 Leu Arg Val Val Ile Pro Pro Ser SerLys Gly Met Met Pro Pro Leu 275 280 285 Ser Glu Glu Glu Glu Leu Glu LeuAsn Thr Gln Arg Ile Ser Ser Ser 290 295 300 Gln Ala Thr Gln Pro Leu AlaThr Pro Val Val Ser Val Thr Thr Pro 305 310 315 320 Ser Leu Pro Pro GlnGly Leu Val Tyr Ser Ala Met Pro Thr Ala Tyr 325 330 335 Asn Thr Asp TyrSer Leu Thr Ser Ala Asp Leu Ser Ala Leu Gln Gly 340 345 350 Phe Asn SerPro Gly Met Leu Ser Leu Gly Gln Val Ser Ala Trp Gln 355 360 365 Gln HisHis Leu Gly Gln Ala Ala Leu Ser Ser Leu Val Ala Gly Gly 370 375 380 GlnLeu Ser Gln Gly Ser Asn Leu Ser Ile Asn Thr Asn Gln Asn Ile 385 390 395400 Ser Ile Lys Ser Glu Pro Ile Ser Pro Pro Arg Asp Arg Met Thr Pro 405410 415 Ser Gly Phe Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Pro420 425 430 Pro Pro Pro Gln Pro Gln Pro Gln Pro Pro Gln Pro Gln Pro ArgGln 435 440 445 Glu Met Gly Arg Ser Pro Val Asp Ser Leu Ser Ser Ser SerSer Ser 450 455 460 Tyr Asp Gly Ser Asp Arg Glu Asp Pro Arg Gly Asp PheHis Ser Pro 465 470 475 480 Ile Val Leu Gly Arg Pro Pro Asn Thr Glu AspArg Glu Ser Pro Ser 485 490 495 Val Lys Arg Met Arg Met Asp Ala Trp ValThr 500 505 14 1521 DNA Homo sapiens CDS (1)..(1524) 14 atg ggg cgg aagaaa ata caa atc aca cgc ata atg gat gaa agg aac 48 Met Gly Arg Lys LysIle Gln Ile Thr Arg Ile Met Asp Glu Arg Asn 1 5 10 15 cga cag gtc actttt aca aag aga aag ttt gga tta atg aag aaa gcc 96 Arg Gln Val Thr PheThr Lys Arg Lys Phe Gly Leu Met Lys Lys Ala 20 25 30 tat gaa ctt agt gtgctc tgt gac tgt gaa ata gca ctc atc att ttc 144 Tyr Glu Leu Ser Val LeuCys Asp Cys Glu Ile Ala Leu Ile Ile Phe 35 40 45 aac agc tct aac aaa ctgttt caa tat gct agc act gat atg gac aaa 192 Asn Ser Ser Asn Lys Leu PheGln Tyr Ala Ser Thr Asp Met Asp Lys 50 55 60 gtt ctt ctc aag tat aca gaatat aat gaa cct cat gaa agc aga acc 240 Val Leu Leu Lys Tyr Thr Glu TyrAsn Glu Pro His Glu Ser Arg Thr 65 70 75 80 aac tcg gat att gtt gag gctctg aac aag aag gaa cac aga ggg tgc 288 Asn Ser Asp Ile Val Glu Ala LeuAsn Lys Lys Glu His Arg Gly Cys 85 90 95 gac agc cca gac cct gat act tcatat gtg cta act cca cat aca gaa 336 Asp Ser Pro Asp Pro Asp Thr Ser TyrVal Leu Thr Pro His Thr Glu 100 105 110 gaa aaa tat aaa aaa att aat gaggaa ttt gat aat atg atg cgg aat 384 Glu Lys Tyr Lys Lys Ile Asn Glu GluPhe Asp Asn Met Met Arg Asn 115 120 125 cat aaa atc gca cct ggt ctg ccacct cag aac ttt tca atg tct gtc 432 His Lys Ile Ala Pro Gly Leu Pro ProGln Asn Phe Ser Met Ser Val 130 135 140 aca gtt cca gtg acc agc ccc aatgct ttg tcc tac act aac cca ggg 480 Thr Val Pro Val Thr Ser Pro Asn AlaLeu Ser Tyr Thr Asn Pro Gly 145 150 155 160 agt tca ctg gtg tcc cca tctttg gca gcc agc tca acg tta aca gat 528 Ser Ser Leu Val Ser Pro Ser LeuAla Ala Ser Ser Thr Leu Thr Asp 165 170 175 tca agc atg ctc tct cca cctcaa acc aca tta cat aga aat gtg tct 576 Ser Ser Met Leu Ser Pro Pro GlnThr Thr Leu His Arg Asn Val Ser 180 185 190 cct gga gct cct cag aga ccacca agt act ggc aat gca ggt ggg atg 624 Pro Gly Ala Pro Gln Arg Pro ProSer Thr Gly Asn Ala Gly Gly Met 195 200 205 ttg agc act aca gac ctc acagtg cca aat gga gct gga agc agt cca 672 Leu Ser Thr Thr Asp Leu Thr ValPro Asn Gly Ala Gly Ser Ser Pro 210 215 220 gtg ggg aat gga ttt gta aactca aga gct tct cca aat ttg att gga 720 Val Gly Asn Gly Phe Val Asn SerArg Ala Ser Pro Asn Leu Ile Gly 225 230 235 240 gct act ggt gca aat agctta ggc aaa gtc atg cct aca aag tct ccc 768 Ala Thr Gly Ala Asn Ser LeuGly Lys Val Met Pro Thr Lys Ser Pro 245 250 255 cct cca cca ggt ggt ggtaat ctt gga atg aac agt agg aaa cca gat 816 Pro Pro Pro Gly Gly Gly AsnLeu Gly Met Asn Ser Arg Lys Pro Asp 260 265 270 ctt cga gtt gtc atc ccccct tca agc aag ggc atg atg cct cca cta 864 Leu Arg Val Val Ile Pro ProSer Ser Lys Gly Met Met Pro Pro Leu 275 280 285 tcg gag gaa gag gaa ttggag ttg aac acc caa agg atc agt agt tct 912 Ser Glu Glu Glu Glu Leu GluLeu Asn Thr Gln Arg Ile Ser Ser Ser 290 295 300 caa gcc act caa cct cttgct acc cca gtc gtg tct gtg aca acc cca 960 Gln Ala Thr Gln Pro Leu AlaThr Pro Val Val Ser Val Thr Thr Pro 305 310 315 320 agc ttg cct ccg caagga ctt gtg tac tca gca atg ccg act gcc tac 1008 Ser Leu Pro Pro Gln GlyLeu Val Tyr Ser Ala Met Pro Thr Ala Tyr 325 330 335 aac act gat tat tcactg acc agc gct gac ctg tca gcc ctt caa ggc 1056 Asn Thr Asp Tyr Ser LeuThr Ser Ala Asp Leu Ser Ala Leu Gln Gly 340 345 350 ttc aac tcg cca ggaatg ctg tcg ctg gga cag gtg tcg gcc tgg cag 1104 Phe Asn Ser Pro Gly MetLeu Ser Leu Gly Gln Val Ser Ala Trp Gln 355 360 365 cag cac cac cta ggacaa gca gcc ctc agc tct ctt gtt gct gga ggg 1152 Gln His His Leu Gly GlnAla Ala Leu Ser Ser Leu Val Ala Gly Gly 370 375 380 cag tta tct cag ggttcc aat tta tcc att aat acc aac caa aac atc 1200 Gln Leu Ser Gln Gly SerAsn Leu Ser Ile Asn Thr Asn Gln Asn Ile 385 390 395 400 agc atc aag tccgaa ccg att tca cct cct cgg gat cgt atg acc cca 1248 Ser Ile Lys Ser GluPro Ile Ser Pro Pro Arg Asp Arg Met Thr Pro 405 410 415 tcg ggc ttc cagcag cag cag cag cag cag cag cag cag cag ccg ccg 1296 Ser Gly Phe Gln GlnGln Gln Gln Gln Gln Gln Gln Gln Gln Pro Pro 420 425 430 cca cca ccg cagccc cag cca caa ccc ccg cag ccc cag ccc cga cag 1344 Pro Pro Pro Gln ProGln Pro Gln Pro Pro Gln Pro Gln Pro Arg Gln 435 440 445 gaa atg ggg cgctcc cct gtg gac agt ctg agc agc tct agt agc tcc 1392 Glu Met Gly Arg SerPro Val Asp Ser Leu Ser Ser Ser Ser Ser Ser 450 455 460 tat gat ggc agtgat cgg gag gat cca cgg ggc gac ttc cat tct cca 1440 Tyr Asp Gly Ser AspArg Glu Asp Pro Arg Gly Asp Phe His Ser Pro 465 470 475 480 att gtg cttggc cga ccc cca aac act gag gac aga gaa agc cct tct 1488 Ile Val Leu GlyArg Pro Pro Asn Thr Glu Asp Arg Glu Ser Pro Ser 485 490 495 gta aag cgaatg agg atg gac gcg tgg gtg acc 1521 Val Lys Arg Met Arg Met Asp Ala TrpVal Thr 500 505 15 365 PRT Homo sapiens 15 Met Gly Arg Lys Lys Ile GlnIle Ser Arg Ile Leu Asp Gln Arg Asn 1 5 10 15 Arg Gln Val Thr Phe ThrLys Arg Lys Phe Gly Leu Met Lys Lys Ala 20 25 30 Tyr Glu Leu Ser Val LeuCys Asp Cys Glu Ile Ala Leu Ile Ile Phe 35 40 45 Asn Ser Ala Asn Arg LeuPhe Gln Tyr Ala Ser Thr Asp Met Asp Arg 50 55 60 Val Leu Leu Lys Tyr ThrGlu Tyr Ser Glu Pro His Glu Ser Arg Thr 65 70 75 80 Asn Thr Asp Ile LeuGlu Thr Leu Lys Arg Arg Gly Ile Gly Leu Asp 85 90 95 Gly Pro Glu Leu GluPro Asp Glu Gly Pro Glu Glu Pro Gly Glu Lys 100 105 110 Phe Arg Arg LeuAla Gly Glu Gly Gly Asp Pro Ala Leu Pro Arg Pro 115 120 125 Arg Leu TyrPro Ala Ala Pro Ala Met Pro Ser Pro Asp Val Val Tyr 130 135 140 Gly AlaLeu Pro Pro Pro Gly Cys Asp Pro Ser Gly Leu Gly Glu Ala 145 150 155 160Leu Pro Ala Gln Ser Arg Pro Ser Pro Phe Arg Pro Ala Ala Pro Lys 165 170175 Ala Gly Pro Pro Gly Leu Val His Pro Leu Phe Ser Pro Ser His Leu 180185 190 Thr Ser Lys Thr Pro Pro Pro Leu Tyr Leu Pro Thr Glu Gly Arg Arg195 200 205 Ser Asp Leu Pro Gly Gly Leu Ala Gly Pro Arg Gly Gly Leu AsnThr 210 215 220 Ser Arg Ser Leu Tyr Ser Gly Leu Gln Asn Pro Cys Ser ThrAla Thr 225 230 235 240 Pro Gly Pro Pro Leu Gly Ser Phe Pro Phe Leu ProGly Gly Pro Pro 245 250 255 Val Gly Ala Glu Ala Trp Ala Arg Arg Val ProGln Pro Ala Ala Pro 260 265 270 Pro Arg Arg Pro Pro Gln Ser Ala Ser SerLeu Ser Ala Ser Leu Arg 275 280 285 Pro Pro Gly Ala Pro Ala Thr Phe LeuArg Pro Ser Pro Ile Pro Cys 290 295 300 Ser Ser Pro Gly Pro Trp Gln SerLeu Cys Gly Leu Gly Pro Pro Cys 305 310 315 320 Ala Gly Cys Pro Trp ProThr Ala Gly Pro Gly Arg Arg Ser Pro Gly 325 330 335 Gly Thr Ser Pro GluArg Ser Pro Gly Thr Ala Arg Ala Arg Gly Asp 340 345 350 Pro Thr Ser LeuGln Ala Ser Ser Glu Lys Thr Gln Gln 355 360 365 16 1095 DNA Homo sapiensCDS (1)..(1098) 16 atg ggg agg aaa aaa atc cag atc tcc cgc atc ctg gaccaa agg aat 48 Met Gly Arg Lys Lys Ile Gln Ile Ser Arg Ile Leu Asp GlnArg Asn 1 5 10 15 cgg cag gtg acg ttc acc aag cgg aag ttc ggg ctg atgaag aag gcc 96 Arg Gln Val Thr Phe Thr Lys Arg Lys Phe Gly Leu Met LysLys Ala 20 25 30 tat gag ctg agc gtg ctc tgt gac tgt gag ata gcc ctc atcatc ttc 144 Tyr Glu Leu Ser Val Leu Cys Asp Cys Glu Ile Ala Leu Ile IlePhe 35 40 45 aac agc gcc aac cgc ctc ttc cag tat gcc agc acg gac atg gaccgt 192 Asn Ser Ala Asn Arg Leu Phe Gln Tyr Ala Ser Thr Asp Met Asp Arg50 55 60 gtg ctg ctg aag tac aca gag tac agc gag ccc cac gag agc cgc acc240 Val Leu Leu Lys Tyr Thr Glu Tyr Ser Glu Pro His Glu Ser Arg Thr 6570 75 80 aac act gac atc ctc gag acg ctg aag cgg agg ggc att ggc ctc gat288 Asn Thr Asp Ile Leu Glu Thr Leu Lys Arg Arg Gly Ile Gly Leu Asp 8590 95 ggg cca gag ctg gag ccg gat gaa ggg cct gag gag cca gga gag aag336 Gly Pro Glu Leu Glu Pro Asp Glu Gly Pro Glu Glu Pro Gly Glu Lys 100105 110 ttt cgg agg ctg gca ggc gaa ggg ggt gat ccg gcc ttg ccc cga ccc384 Phe Arg Arg Leu Ala Gly Glu Gly Gly Asp Pro Ala Leu Pro Arg Pro 115120 125 cgg ctg tat cct gca gct cct gct atg ccc agc cca gat gtg gta tac432 Arg Leu Tyr Pro Ala Ala Pro Ala Met Pro Ser Pro Asp Val Val Tyr 130135 140 ggg gcc tta ccg cca cca ggc tgt gac ccc agt ggg ctt ggg gaa gca480 Gly Ala Leu Pro Pro Pro Gly Cys Asp Pro Ser Gly Leu Gly Glu Ala 145150 155 160 ctg ccc gcc cag agc cgc cca tct ccc ttc cga cca gca gcc cccaaa 528 Leu Pro Ala Gln Ser Arg Pro Ser Pro Phe Arg Pro Ala Ala Pro Lys165 170 175 gcc ggg ccc cca ggc ctg gtg cac cct ctc ttc tca cca agc cacctc 576 Ala Gly Pro Pro Gly Leu Val His Pro Leu Phe Ser Pro Ser His Leu180 185 190 acc agc aag aca cca ccc cca ctg tac ctg ccg acg gaa ggg cggagg 624 Thr Ser Lys Thr Pro Pro Pro Leu Tyr Leu Pro Thr Glu Gly Arg Arg195 200 205 tca gac ctg cct ggt ggc ctg gct ggg ccc cga ggg gga cta aacacc 672 Ser Asp Leu Pro Gly Gly Leu Ala Gly Pro Arg Gly Gly Leu Asn Thr210 215 220 tcc aga agc ctc tac agt ggc ctg cag aac ccc tgc tcc act gcaact 720 Ser Arg Ser Leu Tyr Ser Gly Leu Gln Asn Pro Cys Ser Thr Ala Thr225 230 235 240 ccc gga ccc cca ctg ggg agc ttc ccc ttc ctc ccc gga ggcccc cca 768 Pro Gly Pro Pro Leu Gly Ser Phe Pro Phe Leu Pro Gly Gly ProPro 245 250 255 gtg ggg gcc gaa gcc tgg gcg agg agg gtc ccc caa ccc gcggcg cct 816 Val Gly Ala Glu Ala Trp Ala Arg Arg Val Pro Gln Pro Ala AlaPro 260 265 270 ccc cgc cga ccc ccc cag tca gca tca agt ctg agc gcc tctctc cgg 864 Pro Arg Arg Pro Pro Gln Ser Ala Ser Ser Leu Ser Ala Ser LeuArg 275 280 285 ccc ccg ggg gcc ccg gcg act ttc cta aga cct tcc cct atccct tgc 912 Pro Pro Gly Ala Pro Ala Thr Phe Leu Arg Pro Ser Pro Ile ProCys 290 295 300 tcc tcg ccc ggt ccc tgg cag agc ctc tgc ggc ctg ggc ccgccc tgc 960 Ser Ser Pro Gly Pro Trp Gln Ser Leu Cys Gly Leu Gly Pro ProCys 305 310 315 320 gcc ggc tgc cct tgg ccg acg gct ggc ccc ggt agg agatca ccc ggt 1008 Ala Gly Cys Pro Trp Pro Thr Ala Gly Pro Gly Arg Arg SerPro Gly 325 330 335 ggc acc agc cca gag cgc tcg cca ggt acg gcg agg gcacgt ggg gac 1056 Gly Thr Ser Pro Glu Arg Ser Pro Gly Thr Ala Arg Ala ArgGly Asp 340 345 350 ccc acc tcc ctc cag gcc tct tca gag aag acc caa cag1095 Pro Thr Ser Leu Gln Ala Ser Ser Glu Lys Thr Gln Gln 355 360 365 17465 PRT Homo sapiens 17 Met Gly Arg Lys Lys Ile Gln Ile Thr Arg Ile MetAsp Glu Arg Asn 1 5 10 15 Arg Gln Val Thr Phe Thr Lys Arg Lys Phe GlyLeu Met Lys Lys Ala 20 25 30 Tyr Glu Leu Ser Val Leu Cys Asp Cys Glu IleAla Leu Ile Ile Phe 35 40 45 Asn Ser Thr Asn Lys Leu Phe Gln Tyr Ala SerThr Asp Met Asp Lys 50 55 60 Val Leu Leu Lys Tyr Thr Glu Tyr Asn Glu ProHis Glu Ser Arg Thr 65 70 75 80 Asn Ser Asp Ile Val Glu Thr Leu Arg LysLys Gly Leu Asn Gly Cys 85 90 95 Asp Ser Pro Asp Pro Asp Ala Asp Asp SerVal Gly His Ser Pro Glu 100 105 110 Ser Glu Asp Lys Tyr Arg Lys Ile AsnGlu Asp Ile Asp Leu Met Ile 115 120 125 Ser Arg Gln Arg Leu Cys Ala ValPro Pro Pro Asn Phe Glu Met Pro 130 135 140 Val Ser Ile Pro Val Ser SerHis Asn Ser Leu Val Tyr Ser Asn Pro 145 150 155 160 Val Ser Ser Leu GlyAsn Pro Asn Leu Leu Pro Leu Ala His Pro Ser 165 170 175 Leu Gln Arg AsnSer Met Ser Pro Gly Val Thr His Arg Pro Pro Ser 180 185 190 Ala Gly AsnThr Gly Gly Leu Met Gly Gly Asp Leu Thr Ser Gly Ala 195 200 205 Gly ThrSer Ala Gly Asn Gly Tyr Gly Asn Pro Arg Asn Ser Pro Gly 210 215 220 LeuLeu Val Ser Pro Gly Asn Leu Asn Lys Asn Met Gln Ala Lys Ser 225 230 235240 Pro Pro Pro Met Asn Leu Gly Met Asn Asn Arg Lys Pro Asp Leu Arg 245250 255 Val Leu Ile Pro Pro Gly Ser Lys Asn Thr Met Pro Ser Val Asn Gln260 265 270 Arg Ile Asn Asn Ser Gln Ser Ala Gln Ser Leu Ala Thr Pro ValVal 275 280 285 Ser Val Ala Thr Pro Thr Leu Pro Gly Gln Gly Met Gly GlyTyr Pro 290 295 300 Ser Ala Ile Ser Thr Thr Tyr Gly Thr Glu Tyr Ser LeuSer Ser Ala 305 310 315 320 Asp Leu Ser Ser Leu Ser Gly Phe Asn Thr AlaSer Ala Leu His Leu 325 330 335 Gly Ser Val Thr Gly Trp Gln Gln Gln HisLeu His Asn Met Pro Pro 340 345 350 Ser Ala Leu Ser Gln Leu Gly Ala CysThr Ser Thr His Leu Ser Gln 355 360 365 Ser Ser Asn Leu Ser Leu Pro SerThr Gln Ser Leu Asn Ile Lys Ser 370 375 380 Glu Pro Val Ser Pro Pro ArgAsp Arg Thr Thr Thr Pro Ser Arg Tyr 385 390 395 400 Pro Gln His Thr ArgHis Glu Ala Gly Arg Ser Pro Val Asp Ser Leu 405 410 415 Ser Ser Cys SerSer Ser Tyr Asp Gly Ser Asp Arg Glu Asp His Arg 420 425 430 Asn Glu PheHis Ser Pro Ile Gly Leu Thr Arg Pro Ser Pro Asp Glu 435 440 445 Arg GluSer Pro Ser Val Lys Arg Met Arg Leu Ser Glu Gly Trp Ala 450 455 460 Thr18 1395 DNA Homo sapiens CDS (1)..(1398) 18 atg ggg aga aaa aag att cagatt acg agg att atg gat gaa cgt aac 48 Met Gly Arg Lys Lys Ile Gln IleThr Arg Ile Met Asp Glu Arg Asn 1 5 10 15 aga cag gtg aca ttt aca aagagg aaa ttt ggg ttg atg aag aag gct 96 Arg Gln Val Thr Phe Thr Lys ArgLys Phe Gly Leu Met Lys Lys Ala 20 25 30 tat gag ctg agc gtg ctg tgt gactgt gag att gcg ctg atc atc ttc 144 Tyr Glu Leu Ser Val Leu Cys Asp CysGlu Ile Ala Leu Ile Ile Phe 35 40 45 aac agc acc aac aag ctg ttc cag tatgcc agc acc gac atg gac aaa 192 Asn Ser Thr Asn Lys Leu Phe Gln Tyr AlaSer Thr Asp Met Asp Lys 50 55 60 gtg ctt ctc aag tac acg gag tac aac gagccg cat gag agc cgg aca 240 Val Leu Leu Lys Tyr Thr Glu Tyr Asn Glu ProHis Glu Ser Arg Thr 65 70 75 80 aac tca gac atc gtg gag acg ttg aga aagaag ggc ctt aat ggc tgt 288 Asn Ser Asp Ile Val Glu Thr Leu Arg Lys LysGly Leu Asn Gly Cys 85 90 95 gac agc cca gac ccc gat gcg gac gat tcc gtaggt cac agc cct gag 336 Asp Ser Pro Asp Pro Asp Ala Asp Asp Ser Val GlyHis Ser Pro Glu 100 105 110 tct gag gac aag tac agg aaa att aac gaa gatatt gat cta atg atc 384 Ser Glu Asp Lys Tyr Arg Lys Ile Asn Glu Asp IleAsp Leu Met Ile 115 120 125 agc agg caa aga ttg tgt gct gtt cca cct cccaac ttc gag atg cca 432 Ser Arg Gln Arg Leu Cys Ala Val Pro Pro Pro AsnPhe Glu Met Pro 130 135 140 gtc tcc atc cca gtg tcc agc cac aac agt ttggtg tac agc aac cct 480 Val Ser Ile Pro Val Ser Ser His Asn Ser Leu ValTyr Ser Asn Pro 145 150 155 160 gtc agc tca ctg gga aac ccc aac cta ttgcca ctg gct cac cct tct 528 Val Ser Ser Leu Gly Asn Pro Asn Leu Leu ProLeu Ala His Pro Ser 165 170 175 ctg cag agg aat agt atg tct cct ggt gtaaca cat cga cct cca agt 576 Leu Gln Arg Asn Ser Met Ser Pro Gly Val ThrHis Arg Pro Pro Ser 180 185 190 gca ggt aac aca ggt ggt ctg atg ggt ggagac ctc acg tct ggt gca 624 Ala Gly Asn Thr Gly Gly Leu Met Gly Gly AspLeu Thr Ser Gly Ala 195 200 205 ggc acc agt gca ggg aac ggg tat ggc aatccc cga aac tca cca ggt 672 Gly Thr Ser Ala Gly Asn Gly Tyr Gly Asn ProArg Asn Ser Pro Gly 210 215 220 ctg ctg gtc tca cct ggt aac ttg aac aagaat atg caa gca aaa tct 720 Leu Leu Val Ser Pro Gly Asn Leu Asn Lys AsnMet Gln Ala Lys Ser 225 230 235 240 cct ccc cca atg aat tta gga atg aataac cgt aaa cca gat ctc cga 768 Pro Pro Pro Met Asn Leu Gly Met Asn AsnArg Lys Pro Asp Leu Arg 245 250 255 gtt ctt att cca cca ggc agc aag aatacg atg cca tca gtg aat caa 816 Val Leu Ile Pro Pro Gly Ser Lys Asn ThrMet Pro Ser Val Asn Gln 260 265 270 agg ata aat aac tcc cag tcg gct cagtca ttg gct acc cca gtg gtt 864 Arg Ile Asn Asn Ser Gln Ser Ala Gln SerLeu Ala Thr Pro Val Val 275 280 285 tcc gta gca act cct act tta cca ggacaa gga atg gga gga tat cca 912 Ser Val Ala Thr Pro Thr Leu Pro Gly GlnGly Met Gly Gly Tyr Pro 290 295 300 tca gcc att tca aca aca tat ggt accgag tac tct ctg agt agt gca 960 Ser Ala Ile Ser Thr Thr Tyr Gly Thr GluTyr Ser Leu Ser Ser Ala 305 310 315 320 gac ctg tca tct ctg tct ggg tttaac acc gcc agc gct ctt cac ctt 1008 Asp Leu Ser Ser Leu Ser Gly Phe AsnThr Ala Ser Ala Leu His Leu 325 330 335 ggt tca gta act ggc tgg caa cagcaa cac cta cat aac atg cca cca 1056 Gly Ser Val Thr Gly Trp Gln Gln GlnHis Leu His Asn Met Pro Pro 340 345 350 tct gcc ctc agt cag ttg gga gcttgc act agc act cat tta tct cag 1104 Ser Ala Leu Ser Gln Leu Gly Ala CysThr Ser Thr His Leu Ser Gln 355 360 365 agt tca aat ctc tcc ctg cct tctact caa agc ctc aac atc aag tca 1152 Ser Ser Asn Leu Ser Leu Pro Ser ThrGln Ser Leu Asn Ile Lys Ser 370 375 380 gaa cct gtt tct cct cct aga gaccgt acc acc acc cct tcg aga tac 1200 Glu Pro Val Ser Pro Pro Arg Asp ArgThr Thr Thr Pro Ser Arg Tyr 385 390 395 400 cca caa cac acg cgc cac gaggcg ggg aga tct cct gtt gac agc ttg 1248 Pro Gln His Thr Arg His Glu AlaGly Arg Ser Pro Val Asp Ser Leu 405 410 415 agc agc tgt agc agt tcg tacgac ggg agc gac cga gag gat cac cgg 1296 Ser Ser Cys Ser Ser Ser Tyr AspGly Ser Asp Arg Glu Asp His Arg 420 425 430 aac gaa ttc cac tcc ccc attgga ctc acc aga cct tcg ccg gac gaa 1344 Asn Glu Phe His Ser Pro Ile GlyLeu Thr Arg Pro Ser Pro Asp Glu 435 440 445 agg gaa agt ccc tca gtc aagcgc atg cga ctt tct gaa gga tgg gca 1392 Arg Glu Ser Pro Ser Val Lys ArgMet Arg Leu Ser Glu Gly Trp Ala 450 455 460 aca 1395 Thr 465 19 521 PRTHomo sapiens 19 Met Gly Arg Lys Lys Ile Gln Ile Gln Arg Ile Thr Asp GluArg Asn 1 5 10 15 Arg Gln Val Thr Phe Thr Lys Arg Lys Phe Gly Leu MetLys Lys Ala 20 25 30 Tyr Glu Leu Ser Val Leu Cys Asp Cys Glu Ile Ala LeuIle Ile Phe 35 40 45 Asn His Ser Asn Lys Leu Phe Gln Tyr Ala Ser Thr AspMet Asp Lys 50 55 60 Val Leu Leu Lys Tyr Thr Glu Tyr Asn Glu Pro His GluSer Arg Thr 65 70 75 80 Asn Ala Asp Ile Ile Glu Thr Leu Arg Lys Lys GlyPhe Asn Gly Cys 85 90 95 Asp Ser Pro Glu Pro Asp Gly Glu Asp Ser Leu GluGln Ser Pro Leu 100 105 110 Leu Glu Asp Lys Tyr Arg Arg Ala Ser Glu GluLeu Asp Gly Leu Phe 115 120 125 Arg Arg Tyr Gly Ser Thr Val Pro Ala ProAsn Phe Ala Met Pro Val 130 135 140 Thr Val Pro Val Ser Asn Gln Ser SerLeu Gln Phe Ser Asn Pro Ser 145 150 155 160 Gly Ser Leu Val Thr Pro SerLeu Val Thr Ser Ser Leu Thr Asp Pro 165 170 175 Arg Leu Leu Ser Pro GlnGln Pro Ala Leu Gln Arg Asn Ser Val Ser 180 185 190 Pro Gly Leu Pro GlnArg Pro Ala Ser Ala Gly Ala Met Leu Gly Gly 195 200 205 Asp Leu Asn SerAla Asn Gly Ala Cys Pro Ser Pro Val Gly Asn Gly 210 215 220 Tyr Val SerAla Arg Ala Ser Pro Gly Leu Leu Pro Val Ala Asn Gly 225 230 235 240 AsnSer Leu Asn Lys Val Ile Pro Ala Lys Ser Pro Pro Pro Pro Thr 245 250 255His Ser Thr Gln Leu Gly Ala Pro Ser Arg Lys Pro Asp Leu Arg Val 260 265270 Ile Thr Ser Gln Ala Gly Lys Gly Leu Met His His Leu Thr Glu Asp 275280 285 His Leu Asp Leu Asn Asn Ala Gln Arg Leu Gly Val Ser Gln Ser Thr290 295 300 His Ser Leu Thr Thr Pro Val Val Ser Val Ala Thr Pro Ser LeuLeu 305 310 315 320 Ser Gln Gly Leu Pro Phe Ser Ser Met Pro Thr Ala TyrAsn Thr Asp 325 330 335 Tyr Gln Leu Thr Ser Ala Glu Leu Ser Ser Leu ProAla Phe Ser Ser 340 345 350 Pro Gly Gly Leu Ser Leu Gly Asn Val Thr AlaTrp Gln Gln Pro Gln 355 360 365 Gln Pro Gln Gln Pro Gln Gln Pro Gln ProPro Gln Gln Gln Pro Pro 370 375 380 Gln Pro Gln Gln Pro Gln Pro Gln GlnPro Gln Gln Pro Gln Gln Pro 385 390 395 400 Pro Gln Gln Gln Ser His LeuVal Pro Val Ser Leu Ser Asn Leu Ile 405 410 415 Pro Gly Ser Pro Leu ProHis Val Gly Ala Ala Leu Thr Val Thr Thr 420 425 430 His Pro His Ile SerIle Lys Ser Glu Pro Val Ser Pro Ser Arg Glu 435 440 445 Arg Ser Pro AlaPro Pro Pro Pro Ala Val Phe Pro Ala Ala Arg Pro 450 455 460 Glu Pro GlyAsp Gly Leu Ser Ser Pro Ala Gly Gly Ser Tyr Glu Thr 465 470 475 480 GlyAsp Arg Asp Asp Gly Arg Gly Asp Phe Gly Pro Thr Leu Gly Leu 485 490 495Leu Arg Pro Ala Pro Glu Pro Glu Ala Glu Gly Ser Ala Val Lys Arg 500 505510 Met Arg Leu Asp Thr Trp Thr Leu Lys 515 520 20 1563 DNA Homo sapiensCDS (1)..(1566) 20 atg ggg agg aaa aag att cag atc cag cga atc acc gacgag cgg aac 48 Met Gly Arg Lys Lys Ile Gln Ile Gln Arg Ile Thr Asp GluArg Asn 1 5 10 15 cga cag gtg act ttc acc aag cgg aag ttt ggc ctg atgaag aag gcg 96 Arg Gln Val Thr Phe Thr Lys Arg Lys Phe Gly Leu Met LysLys Ala 20 25 30 tat gag ctg agc gtg cta tgt gac tgc gag atc gca ctc atcatc ttc 144 Tyr Glu Leu Ser Val Leu Cys Asp Cys Glu Ile Ala Leu Ile IlePhe 35 40 45 aac cac tcc aac aag ctg ttc cag tac gcc agc acc gac atg gacaag 192 Asn His Ser Asn Lys Leu Phe Gln Tyr Ala Ser Thr Asp Met Asp Lys50 55 60 gtg ctg ctc aag tac acg gag tac aat gag cca cac gag agc cgc acc240 Val Leu Leu Lys Tyr Thr Glu Tyr Asn Glu Pro His Glu Ser Arg Thr 6570 75 80 aac gcc gac atc atc gag acc ctg agg aag aag ggc ttc aat ggc tgc288 Asn Ala Asp Ile Ile Glu Thr Leu Arg Lys Lys Gly Phe Asn Gly Cys 8590 95 gac agc ccc gag ccc gac ggg gag gac tcg ctg gaa cag agc ccc ctg336 Asp Ser Pro Glu Pro Asp Gly Glu Asp Ser Leu Glu Gln Ser Pro Leu 100105 110 ctg gag gac aag tac cga cgc gcc agc gag gag ctc gac ggg ctc ttc384 Leu Glu Asp Lys Tyr Arg Arg Ala Ser Glu Glu Leu Asp Gly Leu Phe 115120 125 cgg cgc tat ggg tca act gtc ccg gcc ccc aac ttt gcc atg cct gtc432 Arg Arg Tyr Gly Ser Thr Val Pro Ala Pro Asn Phe Ala Met Pro Val 130135 140 acg gtg ccc gtg tcc aat cag agc tca ctg cag ttc agc aat ccc agc480 Thr Val Pro Val Ser Asn Gln Ser Ser Leu Gln Phe Ser Asn Pro Ser 145150 155 160 ggc tcc ctg gtc acc cct tcc ctg gtg aca tca tcc ctc acg gacccg 528 Gly Ser Leu Val Thr Pro Ser Leu Val Thr Ser Ser Leu Thr Asp Pro165 170 175 cgg ctc ctg tcc ccc cag cag cca gca cta cag agg aac agt gtgtct 576 Arg Leu Leu Ser Pro Gln Gln Pro Ala Leu Gln Arg Asn Ser Val Ser180 185 190 cct ggc ctg ccc cag cgg cca gct agt gcg ggg gcc atg ctg gggggt 624 Pro Gly Leu Pro Gln Arg Pro Ala Ser Ala Gly Ala Met Leu Gly Gly195 200 205 gac ctg aac agt gct aac gga gcc tgc ccc agc cct gtt ggg aatggc 672 Asp Leu Asn Ser Ala Asn Gly Ala Cys Pro Ser Pro Val Gly Asn Gly210 215 220 tac gtc agt gct cgg gct tcc cct ggc ctc ctc cct gtg gcc aatggc 720 Tyr Val Ser Ala Arg Ala Ser Pro Gly Leu Leu Pro Val Ala Asn Gly225 230 235 240 aac agc cta aac aag gtc atc cct gcc aag tct ccg ccc ccacct acc 768 Asn Ser Leu Asn Lys Val Ile Pro Ala Lys Ser Pro Pro Pro ProThr 245 250 255 cac agc acc cag ctt gga gcc ccc agc cgc aag ccc gac ctgcga gtc 816 His Ser Thr Gln Leu Gly Ala Pro Ser Arg Lys Pro Asp Leu ArgVal 260 265 270 atc act tcc cag gca gga aag ggg tta atg cat cac ttg actgag gac 864 Ile Thr Ser Gln Ala Gly Lys Gly Leu Met His His Leu Thr GluAsp 275 280 285 cat tta gat ctg aac aat gcc cag cgc ctt ggg gtc tcc cagtct act 912 His Leu Asp Leu Asn Asn Ala Gln Arg Leu Gly Val Ser Gln SerThr 290 295 300 cat tcg ctc acc acc cca gtg gtt tct gtg gca acg ccg agttta ctc 960 His Ser Leu Thr Thr Pro Val Val Ser Val Ala Thr Pro Ser LeuLeu 305 310 315 320 agc cag ggc ctc ccc ttc tct tcc atg ccc act gcc tacaac aca gat 1008 Ser Gln Gly Leu Pro Phe Ser Ser Met Pro Thr Ala Tyr AsnThr Asp 325 330 335 tac cag ttg acc agt gca gag ctc tcc tcc tta cca gccttt agt tca 1056 Tyr Gln Leu Thr Ser Ala Glu Leu Ser Ser Leu Pro Ala PheSer Ser 340 345 350 cct ggg ggg ctg tcg cta ggc aat gtc act gcc tgg caacag cca cag 1104 Pro Gly Gly Leu Ser Leu Gly Asn Val Thr Ala Trp Gln GlnPro Gln 355 360 365 cag ccc cag cag ccg cag cag cca cag cct cca cag cagcag cca ccg 1152 Gln Pro Gln Gln Pro Gln Gln Pro Gln Pro Pro Gln Gln GlnPro Pro 370 375 380 cag cca cag cag cca cag cca cag cag cct cag cag ccgcaa cag cca 1200 Gln Pro Gln Gln Pro Gln Pro Gln Gln Pro Gln Gln Pro GlnGln Pro 385 390 395 400 cct cag caa cag tcc cac ctg gtc cct gta tct ctcagc aac ctc atc 1248 Pro Gln Gln Gln Ser His Leu Val Pro Val Ser Leu SerAsn Leu Ile 405 410 415 ccg ggc agc ccc ctg ccc cac gtg ggt gct gcc ctcaca gtc acc acc 1296 Pro Gly Ser Pro Leu Pro His Val Gly Ala Ala Leu ThrVal Thr Thr 420 425 430 cac ccc cac atc agc atc aag tca gaa ccg gtg tcccca agc cgt gag 1344 His Pro His Ile Ser Ile Lys Ser Glu Pro Val Ser ProSer Arg Glu 435 440 445 cgc agc cct gcg cct ccc cct cca gct gtg ttc ccagct gcc cgc cct 1392 Arg Ser Pro Ala Pro Pro Pro Pro Ala Val Phe Pro AlaAla Arg Pro 450 455 460 gag cct ggc gat ggt ctc agc agc cca gcc ggg ggatcc tat gag acg 1440 Glu Pro Gly Asp Gly Leu Ser Ser Pro Ala Gly Gly SerTyr Glu Thr 465 470 475 480 gga gac cgg gat gac gga cgg ggg gac ttc gggccc aca ctg ggc ctg 1488 Gly Asp Arg Asp Asp Gly Arg Gly Asp Phe Gly ProThr Leu Gly Leu 485 490 495 ctg cgc cca gcc cca gag cct gag gct gag ggctca gct gtg aag agg 1536 Leu Arg Pro Ala Pro Glu Pro Glu Ala Glu Gly SerAla Val Lys Arg 500 505 510 atg cgg ctt gat acc tgg aca tta aag 1563 MetArg Leu Asp Thr Trp Thr Leu Lys 515 520 21 217 PRT Rattus norvegicus 21Met Ser Leu Val Gly Gly Phe Pro His His Pro Val Val His His Glu 1 5 1015 Gly Tyr Pro Phe Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 20 2530 Ser Arg Cys Ser His Glu Glu Asn Pro Tyr Phe His Gly Trp Leu Ile 35 4045 Gly His Pro Glu Met Ser Pro Pro Asp Tyr Ser Met Ala Leu Ser Tyr 50 5560 Ser Pro Glu Tyr Ala Ser Gly Ala Ala Gly Leu Asp His Ser His Tyr 65 7075 80 Gly Gly Val Pro Pro Gly Ala Gly Pro Pro Gly Leu Gly Gly Pro Arg 8590 95 Pro Val Lys Arg Arg Gly Thr Ala Asn Arg Lys Glu Arg Arg Arg Thr100 105 110 Gln Ser Ile Asn Ser Ala Phe Ala Glu Leu Arg Glu Cys Ile ProAsn 115 120 125 Val Pro Ala Asp Thr Lys Leu Ser Lys Ile Lys Thr Leu ArgLeu Ala 130 135 140 Thr Ser Tyr Ile Ala Tyr Leu Met Asp Leu Leu Ala LysAsp Asp Gln 145 150 155 160 Asn Gly Glu Ala Glu Ala Phe Lys Ala Glu IleLys Lys Thr Asp Val 165 170 175 Lys Glu Glu Lys Arg Lys Lys Glu Leu AsnGlu Ile Leu Lys Ser Thr 180 185 190 Val Ser Ser Asn Asp Lys Lys Thr LysGly Arg Thr Gly Trp Pro Gln 195 200 205 His Val Trp Ala Leu Glu Leu LysGln 210 215 22 651 DNA Rattus norvegicus CDS (1)..(654) 22 atg agt ctggtg ggg ggc ttt ccc cac cac ccc gtg gtg cac cat gag 48 Met Ser Leu ValGly Gly Phe Pro His His Pro Val Val His His Glu 1 5 10 15 ggc tac ccgttc gcc gca gcc gca gcc gcc gct gct gct gcc gcc gcc 96 Gly Tyr Pro PheAla Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 20 25 30 agc cgc tgc agtcac gag gag aac ccc tat ttc cac ggc tgg ctt att 144 Ser Arg Cys Ser HisGlu Glu Asn Pro Tyr Phe His Gly Trp Leu Ile 35 40 45 ggc cac ccg gag atgtcg ccc ccc gac tac agc atg gcc ctg tcc tac 192 Gly His Pro Glu Met SerPro Pro Asp Tyr Ser Met Ala Leu Ser Tyr 50 55 60 agt ccc gag tac gcc agcggt gcc gcg ggc ctg gac cac tcc cat tat 240 Ser Pro Glu Tyr Ala Ser GlyAla Ala Gly Leu Asp His Ser His Tyr 65 70 75 80 ggg gga gtg ccg ccc ggtgcc ggg cct ccc ggc ctg ggg ggg ccg cgc 288 Gly Gly Val Pro Pro Gly AlaGly Pro Pro Gly Leu Gly Gly Pro Arg 85 90 95 ccg gtg aag cgt cgg ggc accgcc aac cgc aag gag cgg cgc agg act 336 Pro Val Lys Arg Arg Gly Thr AlaAsn Arg Lys Glu Arg Arg Arg Thr 100 105 110 cag agc atc aac agc gcc ttcgcc gag ctg cgc gag tgc atc ccc aac 384 Gln Ser Ile Asn Ser Ala Phe AlaGlu Leu Arg Glu Cys Ile Pro Asn 115 120 125 gtg ccc gcc gac acc aaa ctctcc aaa atc aag act ctg cgc ctg gcc 432 Val Pro Ala Asp Thr Lys Leu SerLys Ile Lys Thr Leu Arg Leu Ala 130 135 140 acc agc tac atc gcc tac ctcatg gat ctg ctg gcc aag gac gac cag 480 Thr Ser Tyr Ile Ala Tyr Leu MetAsp Leu Leu Ala Lys Asp Asp Gln 145 150 155 160 aac gga gag gcg gag gccttc aag gcg gag atc aag aag acc gac gtg 528 Asn Gly Glu Ala Glu Ala PheLys Ala Glu Ile Lys Lys Thr Asp Val 165 170 175 aaa gag gag aag agg aagaaa gag ctg aat gaa atc ttg aaa agt aca 576 Lys Glu Glu Lys Arg Lys LysGlu Leu Asn Glu Ile Leu Lys Ser Thr 180 185 190 gtg agc agc aac gac aagaaa acc aaa ggc cgg aca ggc tgg cca cag 624 Val Ser Ser Asn Asp Lys LysThr Lys Gly Arg Thr Gly Trp Pro Gln 195 200 205 cac gtc tgg gcc ctg gagctc aag cag 651 His Val Trp Ala Leu Glu Leu Lys Gln 210 215 23 215 PRTHomo sapiens 23 Met Asn Leu Val Gly Ser Tyr Ala His His His His His HisHis Pro 1 5 10 15 His Pro Ala His Pro Met Leu His Glu Pro Phe Leu PheGly Pro Ala 20 25 30 Ser Arg Cys His Gln Glu Arg Pro Tyr Phe Gln Ser TrpLeu Leu Ser 35 40 45 Pro Ala Asp Ala Ala Pro Asp Phe Pro Ala Gly Gly ProPro Pro Ala 50 55 60 Ala Ala Ala Ala Ala Thr Ala Tyr Gly Pro Asp Ala ArgPro Gly Gln 65 70 75 80 Ser Pro Gly Arg Leu Glu Ala Leu Gly Gly Arg LeuGly Arg Arg Lys 85 90 95 Gly Ser Gly Pro Lys Lys Glu Arg Arg Arg Thr GluSer Ile Asn Ser 100 105 110 Ala Phe Ala Glu Leu Arg Glu Cys Ile Pro AsnVal Pro Ala Asp Thr 115 120 125 Lys Leu Ser Lys Ile Lys Thr Leu Arg LeuAla Thr Ser Tyr Ile Ala 130 135 140 Tyr Leu Met Asp Val Leu Ala Lys AspAla Gln Ser Gly Asp Pro Glu 145 150 155 160 Ala Phe Lys Ala Glu Leu LysLys Ala Asp Gly Gly Arg Glu Ser Lys 165 170 175 Arg Lys Arg Glu Leu GlnGln His Glu Gly Phe Pro Pro Ala Leu Gly 180 185 190 Pro Val Glu Lys ArgIle Lys Gly Arg Thr Gly Trp Pro Gln Gln Val 195 200 205 Trp Ala Leu GluLeu Asn Gln 210 215 24 645 DNA Homo sapiens CDS (1)..(648) 24 atg aacctc gtg ggc agc tac gca cac cat cac cac cat cac cac ccg 48 Met Asn LeuVal Gly Ser Tyr Ala His His His His His His His Pro 1 5 10 15 cac cctgcg cac ccc atg ctc cac gaa ccc ttc ctc ttc ggt ccg gcc 96 His Pro AlaHis Pro Met Leu His Glu Pro Phe Leu Phe Gly Pro Ala 20 25 30 tcg cgc tgtcat cag gaa agg ccc tac ttc cag agc tgg ctg ctg agc 144 Ser Arg Cys HisGln Glu Arg Pro Tyr Phe Gln Ser Trp Leu Leu Ser 35 40 45 ccg gct gac gctgcc ccg gac ttc cct gcg ggc ggg ccg ccg ccc gcg 192 Pro Ala Asp Ala AlaPro Asp Phe Pro Ala Gly Gly Pro Pro Pro Ala 50 55 60 gcc gct gca gcc gccacc gcc tat ggt cct gac gcc agg cct ggg cag 240 Ala Ala Ala Ala Ala ThrAla Tyr Gly Pro Asp Ala Arg Pro Gly Gln 65 70 75 80 agc ccc ggg cgg ctggag gcg ctt ggc ggc cgt ctt ggc cgg cgg aaa 288 Ser Pro Gly Arg Leu GluAla Leu Gly Gly Arg Leu Gly Arg Arg Lys 85 90 95 ggc tca gga ccc aag aaggag cgg aga cgc act gag agc att aac agc 336 Gly Ser Gly Pro Lys Lys GluArg Arg Arg Thr Glu Ser Ile Asn Ser 100 105 110 gca ttc gcg gag ttg cgcgag tgc atc ccc aac gtg ccg gcc gac acc 384 Ala Phe Ala Glu Leu Arg GluCys Ile Pro Asn Val Pro Ala Asp Thr 115 120 125 aag ctc tcc aag atc aagact ctg cgc cta gcc acc agc tac atc gcc 432 Lys Leu Ser Lys Ile Lys ThrLeu Arg Leu Ala Thr Ser Tyr Ile Ala 130 135 140 tac ctg atg gac gtg ctggcc aag gat gca cag tct ggc gat ccc gag 480 Tyr Leu Met Asp Val Leu AlaLys Asp Ala Gln Ser Gly Asp Pro Glu 145 150 155 160 gcc ttc aag gct gaactc aag aag gcg gat ggc ggc cgt gag agc aag 528 Ala Phe Lys Ala Glu LeuLys Lys Ala Asp Gly Gly Arg Glu Ser Lys 165 170 175 cgg aaa agg gag ctgcag cag cac gaa ggt ttt cct cct gcc ctg ggc 576 Arg Lys Arg Glu Leu GlnGln His Glu Gly Phe Pro Pro Ala Leu Gly 180 185 190 cca gtc gag aag aggatt aaa gga cgc acc ggc tgg ccg cag caa gtc 624 Pro Val Glu Lys Arg IleLys Gly Arg Thr Gly Trp Pro Gln Gln Val 195 200 205 tgg gcg ctg gag ttaaac cag 645 Trp Ala Leu Glu Leu Asn Gln 210 215 25 411 PRT Homo sapiens25 Met Glu Arg Met Ser Asp Ser Ala Asp Lys Pro Ile Asp Asn Asp Ala 1 510 15 Glu Gly Val Trp Ser Pro Asp Ile Glu Gln Ser Phe Gln Glu Ala Leu 2025 30 Ala Ile Tyr Pro Pro Cys Gly Arg Arg Lys Ile Ile Leu Ser Asp Glu 3540 45 Gly Lys Met Tyr Gly Arg Asn Glu Leu Ile Ala Arg Tyr Ile Lys Leu 5055 60 Arg Thr Gly Lys Thr Arg Thr Arg Lys Gln Val Ser Ser His Ile Gln 6570 75 80 Val Leu Ala Arg Arg Lys Ser Arg Asp Phe His Ser Lys Leu Lys Asp85 90 95 Gln Thr Ala Lys Asp Lys Ala Leu Gln His Met Ala Ala Met Ser Ser100 105 110 Ala Gln Ile Val Ser Ala Thr Ala Ile His Asn Lys Leu Gly LeuPro 115 120 125 Gly Ile Pro Arg Pro Thr Phe Pro Gly Ala Pro Gly Phe TrpPro Gly 130 135 140 Met Ile Gln Thr Gly Gln Pro Gly Ser Ser Gln Asp ValLys Pro Phe 145 150 155 160 Val Gln Gln Ala Tyr Pro Ile Gln Pro Ala ValThr Ala Pro Ile Pro 165 170 175 Gly Phe Glu Pro Ala Ser Ala Pro Ala ProSer Val Pro Ala Trp Gln 180 185 190 Gly Arg Ser Ile Gly Thr Thr Lys LeuArg Leu Val Glu Phe Ser Ala 195 200 205 Phe Leu Glu Gln Gln Arg Asp ProAsp Ser Tyr Asn Lys His Leu Phe 210 215 220 Val His Ile Gly His Ala AsnHis Ser Tyr Ser Asp Pro Leu Leu Glu 225 230 235 240 Ser Val Asp Ile ArgGln Ile Tyr Asp Lys Phe Pro Glu Lys Lys Gly 245 250 255 Gly Leu Lys GluLeu Phe Gly Lys Gly Pro Gln Asn Ala Phe Phe Leu 260 265 270 Val Lys PheTrp Ala Asp Leu Asn Cys Asn Ile Gln Asp Asp Ala Gly 275 280 285 Ala PheTyr Gly Val Thr Ser Gln Tyr Glu Ser Ser Glu Asn Met Thr 290 295 300 ValThr Cys Ser Thr Lys Val Cys Ser Phe Gly Lys Gln Val Val Glu 305 310 315320 Lys Val Glu Thr Glu Tyr Ala Arg Phe Glu Asn Gly Arg Phe Val Tyr 325330 335 Arg Ile Asn Arg Ser Pro Met Cys Glu Tyr Met Ile Asn Phe Ile His340 345 350 Lys Leu Lys His Leu Pro Glu Lys Tyr Met Met Asn Ser Val LeuGlu 355 360 365 Asn Phe Thr Ile Leu Leu Val Val Thr Asn Arg Asp Thr GlnGlu Thr 370 375 380 Leu Leu Cys Met Ala Cys Val Phe Glu Val Ser Asn SerGlu His Gly 385 390 395 400 Ala Gln His His Ile Tyr Arg Leu Val Lys Asp405 410 26 1233 DNA Homo sapiens CDS (1)..(1236) 26 atg gaa agg atg agtgac tct gca gat aag cca att gac aat gat gca 48 Met Glu Arg Met Ser AspSer Ala Asp Lys Pro Ile Asp Asn Asp Ala 1 5 10 15 gaa ggg gtc tgg agcccc gac atc gag caa agc ttt cag gag gcc ctg 96 Glu Gly Val Trp Ser ProAsp Ile Glu Gln Ser Phe Gln Glu Ala Leu 20 25 30 gct atc tat cca cca tgtggg agg agg aaa atc atc tta tca gac gaa 144 Ala Ile Tyr Pro Pro Cys GlyArg Arg Lys Ile Ile Leu Ser Asp Glu 35 40 45 ggc aaa atg tat ggt agg aatgaa ttg ata gcc aga tac atc aaa ctc 192 Gly Lys Met Tyr Gly Arg Asn GluLeu Ile Ala Arg Tyr Ile Lys Leu 50 55 60 agg aca ggc aag acg agg acc agaaaa cag gtg tct agt cac att cag 240 Arg Thr Gly Lys Thr Arg Thr Arg LysGln Val Ser Ser His Ile Gln 65 70 75 80 gtt ctt gcc aga agg aaa tct cgtgat ttt cat tcc aag cta aag gat 288 Val Leu Ala Arg Arg Lys Ser Arg AspPhe His Ser Lys Leu Lys Asp 85 90 95 cag act gca aag gat aag gcc ctg cagcac atg gcg gcc atg tcc tca 336 Gln Thr Ala Lys Asp Lys Ala Leu Gln HisMet Ala Ala Met Ser Ser 100 105 110 gcc cag atc gtc tcg gcc act gcc attcat aac aag ctg ggg ctg cct 384 Ala Gln Ile Val Ser Ala Thr Ala Ile HisAsn Lys Leu Gly Leu Pro 115 120 125 ggg att cca cgc ccg acc ttc cca ggggcg ccg ggg ttc tgg ccg gga 432 Gly Ile Pro Arg Pro Thr Phe Pro Gly AlaPro Gly Phe Trp Pro Gly 130 135 140 atg att caa aca ggg cag cca gga tcctca caa gac gtc aag cct ttt 480 Met Ile Gln Thr Gly Gln Pro Gly Ser SerGln Asp Val Lys Pro Phe 145 150 155 160 gtg cag cag gcc tac ccc atc cagcca gcg gtc aca gcc ccc att cca 528 Val Gln Gln Ala Tyr Pro Ile Gln ProAla Val Thr Ala Pro Ile Pro 165 170 175 ggg ttt gag cct gca tcg gcc ccagct ccc tca gtc cct gcc tgg caa 576 Gly Phe Glu Pro Ala Ser Ala Pro AlaPro Ser Val Pro Ala Trp Gln 180 185 190 ggt cgc tcc att ggc aca acc aagctt cgc ctg gtg gaa ttt tca gct 624 Gly Arg Ser Ile Gly Thr Thr Lys LeuArg Leu Val Glu Phe Ser Ala 195 200 205 ttt ctc gag cag cag cga gac ccagac tcg tac aac aaa cac ctc ttc 672 Phe Leu Glu Gln Gln Arg Asp Pro AspSer Tyr Asn Lys His Leu Phe 210 215 220 gtg cac att ggg cat gcc aac cattct tac agt gac cca ttg ctt gaa 720 Val His Ile Gly His Ala Asn His SerTyr Ser Asp Pro Leu Leu Glu 225 230 235 240 tca gtg gac att cgt cag atttat gac aaa ttt cct gaa aag aaa ggt 768 Ser Val Asp Ile Arg Gln Ile TyrAsp Lys Phe Pro Glu Lys Lys Gly 245 250 255 ggc tta aag gaa ctg ttt ggaaag ggc cct caa aat gcc ttc ttc ctc 816 Gly Leu Lys Glu Leu Phe Gly LysGly Pro Gln Asn Ala Phe Phe Leu 260 265 270 gta aaa ttc tgg gct gat ttaaac tgc aat att caa gat gat gct ggg 864 Val Lys Phe Trp Ala Asp Leu AsnCys Asn Ile Gln Asp Asp Ala Gly 275 280 285 gct ttt tat ggt gta acc agtcag tac gag agt tct gaa aat atg aca 912 Ala Phe Tyr Gly Val Thr Ser GlnTyr Glu Ser Ser Glu Asn Met Thr 290 295 300 gtc acc tgt tcc acc aaa gtttgc tcc ttt ggg aag caa gta gta gaa 960 Val Thr Cys Ser Thr Lys Val CysSer Phe Gly Lys Gln Val Val Glu 305 310 315 320 aaa gta gag acg gag tatgca agg ttt gag aat ggc cga ttt gta tac 1008 Lys Val Glu Thr Glu Tyr AlaArg Phe Glu Asn Gly Arg Phe Val Tyr 325 330 335 cga ata aac cgc tcc ccaatg tgt gaa tat atg atc aac ttc atc cac 1056 Arg Ile Asn Arg Ser Pro MetCys Glu Tyr Met Ile Asn Phe Ile His 340 345 350 aag ctc aaa cac tta ccagag aaa tat atg atg aac agt gtt ttg gaa 1104 Lys Leu Lys His Leu Pro GluLys Tyr Met Met Asn Ser Val Leu Glu 355 360 365 aac ttc aca att tta ttggtg gta aca aac agg gat aca caa gaa act 1152 Asn Phe Thr Ile Leu Leu ValVal Thr Asn Arg Asp Thr Gln Glu Thr 370 375 380 cta ctc tgc atg gcc tgtgtg ttt gaa gtt tca aat agt gaa cac gga 1200 Leu Leu Cys Met Ala Cys ValPhe Glu Val Ser Asn Ser Glu His Gly 385 390 395 400 gca caa cat cat atttac agg ctt gta aag gac 1233 Ala Gln His His Ile Tyr Arg Leu Val Lys Asp405 410 27 427 PRT Homo sapiens 27 Ile Thr Ser Asn Glu Trp Ser Ser ProThr Ser Pro Glu Gly Ser Thr 1 5 10 15 Ala Ser Gly Gly Ser Gln Ala LeuAsp Lys Pro Ile Asp Asn Asp Ala 20 25 30 Glu Gly Val Trp Ser Pro Asp IleGlu Gln Ser Phe Gln Glu Ala Leu 35 40 45 Ala Ile Tyr Pro Pro Cys Gly ArgArg Lys Ile Ile Leu Ser Asp Glu 50 55 60 Gly Lys Met Tyr Gly Arg Asn GluLeu Ile Ala Arg Tyr Ile Lys Leu 65 70 75 80 Arg Thr Gly Lys Thr Arg ThrArg Lys Gln Val Ser Ser His Ile Gln 85 90 95 Val Leu Ala Arg Arg Lys AlaArg Glu Ile Gln Ala Lys Leu Lys Asp 100 105 110 Gln Ala Ala Lys Asp LysAla Leu Gln Ser Met Ala Ala Met Ser Ser 115 120 125 Ala Gln Ile Ile SerAla Thr Ala Phe His Ser Ser Met Ala Leu Ala 130 135 140 Arg Gly Pro GlyArg Pro Ala Val Ser Gly Phe Trp Gln Gly Ala Leu 145 150 155 160 Pro GlyGln Ala Gly Thr Ser His Asp Val Lys Pro Phe Ser Gln Gln 165 170 175 ThrTyr Ala Val Gln Pro Pro Leu Pro Leu Pro Gly Phe Glu Ser Pro 180 185 190Ala Gly Pro Ala Pro Ser Pro Ser Ala Pro Pro Ala Pro Pro Trp Gln 195 200205 Gly Arg Ser Val Ala Ser Ser Lys Leu Trp Met Leu Glu Phe Ser Ala 210215 220 Phe Leu Glu Gln Gln Gln Asp Pro Asp Thr Tyr Asn Lys His Leu Phe225 230 235 240 Val His Ile Gly Gln Ser Ser Pro Ser Tyr Ser Asp Pro TyrLeu Glu 245 250 255 Ala Val Asp Ile Arg Gln Ile Tyr Asp Lys Phe Pro GluLys Lys Gly 260 265 270 Gly Leu Lys Asp Leu Phe Glu Arg Gly Pro Ser AsnAla Phe Phe Leu 275 280 285 Val Lys Phe Trp Ala Asp Leu Asn Thr Asn IleGlu Asp Glu Gly Ser 290 295 300 Ser Phe Tyr Gly Val Ser Ser Gln Tyr GluSer Pro Glu Asn Met Ile 305 310 315 320 Ile Thr Cys Ser Thr Lys Val CysSer Phe Gly Lys Gln Val Val Glu 325 330 335 Lys Val Glu Thr Glu Tyr AlaArg Tyr Glu Asn Gly His Tyr Ser Tyr 340 345 350 Arg Ile His Arg Ser ProLeu Cys Glu Tyr Met Ile Asn Phe Ile His 355 360 365 Lys Leu Lys His LeuPro Glu Lys Tyr Met Met Asn Ser Val Leu Glu 370 375 380 Asn Phe Thr IleLeu Gln Val Val Thr Asn Arg Asp Thr Gln Glu Thr 385 390 395 400 Leu LeuCys Ile Ala Tyr Val Phe Glu Val Ser Ala Ser Glu His Gly 405 410 415 AlaGln His His Ile Tyr Arg Leu Val Lys Glu 420 425 28 1281 DNA Homo sapiensCDS (1)..(1284) 28 att acc tcc aac gag tgg agc tct ccc acc tcc cct gagggg agc acc 48 Ile Thr Ser Asn Glu Trp Ser Ser Pro Thr Ser Pro Glu GlySer Thr 1 5 10 15 gcc tct ggg ggc agt cag gca ctg gac aag ccc atc gacaat gac gca 96 Ala Ser Gly Gly Ser Gln Ala Leu Asp Lys Pro Ile Asp AsnAsp Ala 20 25 30 gag ggc gtg tgg agc ccg gat att gag cag agt ttc cag gaggcc ctc 144 Glu Gly Val Trp Ser Pro Asp Ile Glu Gln Ser Phe Gln Glu AlaLeu 35 40 45 gcc atc tac ccg ccc tgt ggc agg cgc aaa atc atc ctg tcg gacgag 192 Ala Ile Tyr Pro Pro Cys Gly Arg Arg Lys Ile Ile Leu Ser Asp Glu50 55 60 ggc aag atg tat ggt cgg aac gag ctg att gcc cgc tac atc aag ctc240 Gly Lys Met Tyr Gly Arg Asn Glu Leu Ile Ala Arg Tyr Ile Lys Leu 6570 75 80 cgg aca ggg aag acc cgc acc agg aag cag gtc tcc agc cac atc cag288 Arg Thr Gly Lys Thr Arg Thr Arg Lys Gln Val Ser Ser His Ile Gln 8590 95 gtg ctg gct cgt cgc aaa gct cgc gag atc cag gcc aag cta aag gac336 Val Leu Ala Arg Arg Lys Ala Arg Glu Ile Gln Ala Lys Leu Lys Asp 100105 110 cag gca gct aag gac aag gcc ctg cag agc atg gct gcc atg tcg tct384 Gln Ala Ala Lys Asp Lys Ala Leu Gln Ser Met Ala Ala Met Ser Ser 115120 125 gca cag atc atc tcc gcc acg gcc ttc cac agt agc atg gcc ctc gcc432 Ala Gln Ile Ile Ser Ala Thr Ala Phe His Ser Ser Met Ala Leu Ala 130135 140 cgg ggc ccc ggc cgc cca gca gtc tca ggg ttt tgg caa gga gct ttg480 Arg Gly Pro Gly Arg Pro Ala Val Ser Gly Phe Trp Gln Gly Ala Leu 145150 155 160 cca ggc caa gcc gga acg tcc cat gat gtg aag cct ttc tct cagcaa 528 Pro Gly Gln Ala Gly Thr Ser His Asp Val Lys Pro Phe Ser Gln Gln165 170 175 acc tat gct gtc cag cct ccg ctg cct ctg cca ggg ttt gag tctcct 576 Thr Tyr Ala Val Gln Pro Pro Leu Pro Leu Pro Gly Phe Glu Ser Pro180 185 190 gca ggg ccc gcc cca tcg ccc tct gcg ccc ccg gca ccc cca tggcag 624 Ala Gly Pro Ala Pro Ser Pro Ser Ala Pro Pro Ala Pro Pro Trp Gln195 200 205 ggc cgc agc gtg gcc agc tcc aag ctc tgg atg ttg gag ttc tctgcc 672 Gly Arg Ser Val Ala Ser Ser Lys Leu Trp Met Leu Glu Phe Ser Ala210 215 220 ttc ctg gag cag cag cag gac ccg gac acg tac aac aag cac ctgttc 720 Phe Leu Glu Gln Gln Gln Asp Pro Asp Thr Tyr Asn Lys His Leu Phe225 230 235 240 gtg cac att ggc cag tcc agc cca agc tac agc gac ccc tacctc gaa 768 Val His Ile Gly Gln Ser Ser Pro Ser Tyr Ser Asp Pro Tyr LeuGlu 245 250 255 gcc gtg gac atc cgc caa atc tat gac aaa ttc ccg gag aaaaag ggt 816 Ala Val Asp Ile Arg Gln Ile Tyr Asp Lys Phe Pro Glu Lys LysGly 260 265 270 gga ctc aag gat ctc ttc gaa cgg gga ccc tcc aat gcc tttttt ctt 864 Gly Leu Lys Asp Leu Phe Glu Arg Gly Pro Ser Asn Ala Phe PheLeu 275 280 285 gtg aag ttc tgg gca gac ctc aac acc aac atc gag gat gaaggc agc 912 Val Lys Phe Trp Ala Asp Leu Asn Thr Asn Ile Glu Asp Glu GlySer 290 295 300 tcc ttc tat ggg gtc tcc agc cag tat gag agc ccc gag aacatg atc 960 Ser Phe Tyr Gly Val Ser Ser Gln Tyr Glu Ser Pro Glu Asn MetIle 305 310 315 320 atc acc tgc tcc acg aag gtc tgc tct ttc ggc aag caggtg gtg gag 1008 Ile Thr Cys Ser Thr Lys Val Cys Ser Phe Gly Lys Gln ValVal Glu 325 330 335 aaa gtt gag aca gag tat gct cgc tat gag aat gga cactac tct tac 1056 Lys Val Glu Thr Glu Tyr Ala Arg Tyr Glu Asn Gly His TyrSer Tyr 340 345 350 cgc atc cac cgg tcc ccg ctc tgt gag tac atg atc aacttc atc cac 1104 Arg Ile His Arg Ser Pro Leu Cys Glu Tyr Met Ile Asn PheIle His 355 360 365 aag ctc aag cac ctc cct gag aag tac atg atg aac agcgtg ctg gag 1152 Lys Leu Lys His Leu Pro Glu Lys Tyr Met Met Asn Ser ValLeu Glu 370 375 380 aac ttc acc atc ctg cag gtg gtc acc aac aga gac acacag gag acc 1200 Asn Phe Thr Ile Leu Gln Val Val Thr Asn Arg Asp Thr GlnGlu Thr 385 390 395 400 ttg ctg tgc att gcc tat gtc ttt gag gtg tca gccagt gag cac ggg 1248 Leu Leu Cys Ile Ala Tyr Val Phe Glu Val Ser Ala SerGlu His Gly 405 410 415 gct cag cac cac atc tac agg ctg gtg aaa gaa 1281Ala Gln His His Ile Tyr Arg Leu Val Lys Glu 420 425 29 435 PRT Homosapiens 29 Ile Ala Ser Asn Ser Trp Asn Ala Ser Ser Ser Pro Gly Glu AlaArg 1 5 10 15 Glu Asp Gly Pro Glu Gly Leu Asp Lys Gly Leu Asp Asn AspAla Glu 20 25 30 Gly Val Trp Ser Pro Asp Ile Glu Gln Ser Phe Gln Glu AlaLeu Ala 35 40 45 Ile Tyr Pro Pro Cys Gly Arg Arg Lys Ile Ile Leu Ser AspGlu Gly 50 55 60 Lys Met Tyr Gly Arg Asn Glu Leu Ile Ala Arg Tyr Ile LysLeu Arg 65 70 75 80 Thr Gly Lys Thr Arg Thr Arg Lys Gln Val Ser Ser HisIle Gln Val 85 90 95 Leu Ala Arg Lys Lys Val Arg Glu Tyr Gln Val Gly IleLys Ala Met 100 105 110 Asn Leu Asp Gln Val Ser Lys Asp Lys Ala Leu GlnSer Met Ala Ser 115 120 125 Met Ser Ser Ala Gln Ile Val Ser Ala Ser ValLeu Gln Asn Lys Phe 130 135 140 Ser Pro Pro Ser Pro Leu Pro Gln Ala ValPhe Ser Thr Ser Ser Arg 145 150 155 160 Phe Trp Ser Ser Pro Pro Leu LeuGly Gln Gln Pro Gly Pro Ser Gln 165 170 175 Asp Ile Lys Pro Phe Ala GlnPro Ala Tyr Pro Ile Gln Pro Pro Leu 180 185 190 Pro Pro Thr Leu Ser SerTyr Glu Pro Leu Ala Pro Leu Pro Ser Ala 195 200 205 Ala Ala Ser Val ProVal Trp Gln Asp Arg Thr Ile Ala Ser Ser Arg 210 215 220 Leu Arg Leu LeuGlu Tyr Ser Ala Phe Met Glu Val Gln Arg Asp Pro 225 230 235 240 Asp ThrTyr Ser Lys His Leu Phe Val His Ile Gly Gln Thr Asn Pro 245 250 255 AlaPhe Ser Asp Pro Pro Leu Glu Ala Val Asp Val Arg Gln Ile Tyr 260 265 270Asp Lys Phe Pro Glu Lys Lys Gly Gly Leu Lys Glu Leu Tyr Glu Lys 275 280285 Gly Pro Pro Asn Ala Phe Phe Leu Val Lys Phe Trp Ala Asp Leu Asn 290295 300 Ser Thr Ile Gln Glu Gly Pro Gly Ala Phe Tyr Gly Val Ser Ser Gln305 310 315 320 Tyr Ser Ser Ala Asp Ser Met Thr Ile Ser Val Ser Thr LysVal Cys 325 330 335 Ser Phe Gly Lys Gln Val Val Glu Lys Val Glu Thr GluTyr Ala Arg 340 345 350 Leu Glu Asn Gly Arg Phe Val Tyr Arg Ile His ArgSer Pro Met Cys 355 360 365 Glu Tyr Met Ile Asn Phe Ile His Lys Leu LysHis Leu Pro Glu Lys 370 375 380 Tyr Met Met Asn Ser Val Leu Glu Asn PheThr Ile Leu Gln Val Val 385 390 395 400 Thr Ser Arg Asp Ser Gln Glu ThrLeu Leu Val Ile Ala Phe Val Phe 405 410 415 Glu Val Ser Thr Ser Glu HisGly Ala Gln His His Val Tyr Lys Leu 420 425 430 Val Lys Asp 30 1305 DNAHomo sapiens CDS (1)..(1305) 30 ata gcg tcc aac agc tgg aac gcc agc agcagc ccc ggg gag gcc cgg 48 Ile Ala Ser Asn Ser Trp Asn Ala Ser Ser SerPro Gly Glu Ala Arg 1 5 10 15 gag gat ggg ccc gag ggc ctg gac aag gggctg gac aac gat gcg gag 96 Glu Asp Gly Pro Glu Gly Leu Asp Lys Gly LeuAsp Asn Asp Ala Glu 20 25 30 ggc gtg tgg agc ccg gac atc gag cag agc ttccag gag gcc ctg gcc 144 Gly Val Trp Ser Pro Asp Ile Glu Gln Ser Phe GlnGlu Ala Leu Ala 35 40 45 atc tac ccg ccc tgc ggc cgg cgg aag atc atc ctgtca gac gag ggc 192 Ile Tyr Pro Pro Cys Gly Arg Arg Lys Ile Ile Leu SerAsp Glu Gly 50 55 60 aag atg tac ggc cga aat gag ttg att gca cgc tat attaaa ctg agg 240 Lys Met Tyr Gly Arg Asn Glu Leu Ile Ala Arg Tyr Ile LysLeu Arg 65 70 75 80 acg ggg aag act cgg acg aga aaa cag gtg tcc agc cacata cag gtt 288 Thr Gly Lys Thr Arg Thr Arg Lys Gln Val Ser Ser His IleGln Val 85 90 95 cta gct cgg aag aag gtg cgg gag tac cag gtt ggc atc aaggcc atg 336 Leu Ala Arg Lys Lys Val Arg Glu Tyr Gln Val Gly Ile Lys AlaMet 100 105 110 aac ctg gac cag gtc tcc aag gac aaa gcc ctt cag agc atggcg tcc 384 Asn Leu Asp Gln Val Ser Lys Asp Lys Ala Leu Gln Ser Met AlaSer 115 120 125 atg tcc tct gcc cag atc gtc tct gcc agt gtc ctg cag aacaag ttc 432 Met Ser Ser Ala Gln Ile Val Ser Ala Ser Val Leu Gln Asn LysPhe 130 135 140 agc cca cct tcc cct ctg ccc cag gcc gtc ttc tcc act tcctcg cgg 480 Ser Pro Pro Ser Pro Leu Pro Gln Ala Val Phe Ser Thr Ser SerArg 145 150 155 160 ttc tgg agc agc ccc cct ctc ctg gga cag cag cct ggaccc tct cag 528 Phe Trp Ser Ser Pro Pro Leu Leu Gly Gln Gln Pro Gly ProSer Gln 165 170 175 gac atc aag ccc ttt gca cag cca gcc tac ccc atc cagccg ccc ctg 576 Asp Ile Lys Pro Phe Ala Gln Pro Ala Tyr Pro Ile Gln ProPro Leu 180 185 190 ccg ccg acg ctc agc agt tat gag ccc ctg gcc ccg ctcccc tca gct 624 Pro Pro Thr Leu Ser Ser Tyr Glu Pro Leu Ala Pro Leu ProSer Ala 195 200 205 gct gcc tct gtg cct gtg tgg cag gac cgt acc att gcctcc tcc cgg 672 Ala Ala Ser Val Pro Val Trp Gln Asp Arg Thr Ile Ala SerSer Arg 210 215 220 ctg cgg ctc ctg gag tat tca gcc ttc atg gag gtg cagcga gac cct 720 Leu Arg Leu Leu Glu Tyr Ser Ala Phe Met Glu Val Gln ArgAsp Pro 225 230 235 240 gac acg tac agc aaa cac ctg ttt gtg cac atc ggccag acg aac ccc 768 Asp Thr Tyr Ser Lys His Leu Phe Val His Ile Gly GlnThr Asn Pro 245 250 255 gcc ttc tca gac cca ccc ctg gag gca gta gat gtgcgc cag atc tat 816 Ala Phe Ser Asp Pro Pro Leu Glu Ala Val Asp Val ArgGln Ile Tyr 260 265 270 gac aaa ttc ccc gag aaa aag gga gga ttg aag gagctc tat gag aag 864 Asp Lys Phe Pro Glu Lys Lys Gly Gly Leu Lys Glu LeuTyr Glu Lys 275 280 285 ggg ccc cct aat gcc ttc ttc ctt gtc aag ttc tgggcc gac ctc aac 912 Gly Pro Pro Asn Ala Phe Phe Leu Val Lys Phe Trp AlaAsp Leu Asn 290 295 300 agc acc atc cag gag ggc ccg gga gcc ttc tat ggggtc agc tct cag 960 Ser Thr Ile Gln Glu Gly Pro Gly Ala Phe Tyr Gly ValSer Ser Gln 305 310 315 320 tac agc tct gct gat agc atg acc atc agc gtctcc acc aag gtg tgc 1008 Tyr Ser Ser Ala Asp Ser Met Thr Ile Ser Val SerThr Lys Val Cys 325 330 335 tcc ttt ggc aaa cag gtg gta gag aag gtg gagact gag tat gcc agg 1056 Ser Phe Gly Lys Gln Val Val Glu Lys Val Glu ThrGlu Tyr Ala Arg 340 345 350 ctg gag aac ggg cgc ttt gtg tac cgt atc caccgc tcg ccc atg tgc 1104 Leu Glu Asn Gly Arg Phe Val Tyr Arg Ile His ArgSer Pro Met Cys 355 360 365 gag tac atg atc aac ttc atc cac aag ctg aagcac ctg ccc gag aag 1152 Glu Tyr Met Ile Asn Phe Ile His Lys Leu Lys HisLeu Pro Glu Lys 370 375 380 tac atg atg aac agc gtg ctg gag aac ttc accatc ctg cag gtg gtc 1200 Tyr Met Met Asn Ser Val Leu Glu Asn Phe Thr IleLeu Gln Val Val 385 390 395 400 acg agc cgg gac tcc cag gag acc ttg cttgtc att gct ttt gtc ttc 1248 Thr Ser Arg Asp Ser Gln Glu Thr Leu Leu ValIle Ala Phe Val Phe 405 410 415 gaa gtc tcc acc agt gag cac ggg gcc cagcac cat gtc tac aag ctc 1296 Glu Val Ser Thr Ser Glu His Gly Ala Gln HisHis Val Tyr Lys Leu 420 425 430 gtc aaa gac 1305 Val Lys Asp 435 31 1132PRT Homo sapiens 31 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser LeuLeu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe ValArg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp ProAla Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro TrpAsp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser CysLeu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu ArgGly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly AlaArg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser TyrLeu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala TrpGly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His LeuLeu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser CysAla Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala AlaThr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg ArgLeu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala GlyVal Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly GlySer Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln HisHis 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp AspThr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe LeuTyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu LeuSer Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val GluThr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro ArgArg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro LeuPhe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr GlyVal Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr ProAla Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val AlaAla Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln LeuLeu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val ArgAla Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala LysPhe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu ArgSer Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn ArgLeu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser IleGly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu SerGlu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu LeuThr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg ProIle Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe ArgArg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala LeuPhe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu LeuGly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp ArgThr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu LeuTyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu ThrSer 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser LeuAsn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg PheMet Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val GlnCys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu CysSer Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly IleArg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu LeuVal Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg ThrLeu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu ArgLys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly GlyThr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp CysGly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp TyrSer Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe HisGln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val IleSer Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys AlaLys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala GlyPro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln AlaPhe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val ProLeu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 ArgLys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 11101115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 323396 DNA Homo sapiens CDS (1)..(3399) 32 atg ccg cgc gct ccc cgc tgc cgagcc gtg cgc tcc ctg ctg cgc agc 48 Met Pro Arg Ala Pro Arg Cys Arg AlaVal Arg Ser Leu Leu Arg Ser 1 5 10 15 cac tac cgc gag gtg ctg ccg ctggcc acg ttc gtg cgg cgc ctg ggg 96 His Tyr Arg Glu Val Leu Pro Leu AlaThr Phe Val Arg Arg Leu Gly 20 25 30 ccc cag ggc tgg cgg ctg gtg cag cgcggg gac ccg gcg gct ttc cgc 144 Pro Gln Gly Trp Arg Leu Val Gln Arg GlyAsp Pro Ala Ala Phe Arg 35 40 45 gcg ctg gtg gcc cag tgc ctg gtg tgc gtgccc tgg gac gca cgg ccg 192 Ala Leu Val Ala Gln Cys Leu Val Cys Val ProTrp Asp Ala Arg Pro 50 55 60 ccc ccc gcc gcc ccc tcc ttc cgc cag gtg tcctgc ctg aag gag ctg 240 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser CysLeu Lys Glu Leu 65 70 75 80 gtg gcc cga gtg ctg cag agg ctg tgc gag cgcggc gcg aag aac gtg 288 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg GlyAla Lys Asn Val 85 90 95 ctg gcc ttc ggc ttc gcg ctg ctg gac ggg gcc cgcggg ggc ccc ccc 336 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg GlyGly Pro Pro 100 105 110 gag gcc ttc acc acc agc gtg cgc agc tac ctg cccaac acg gtg acc 384 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro AsnThr Val Thr 115 120 125 gac gca ctg cgg ggg agc ggg gcg tgg ggg ctg ctgctg cgc cgc gtg 432 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu LeuArg Arg Val 130 135 140 ggc gac gac gtg ctg gtt cac ctg ctg gca cgc tgcgcg ctc ttt gtg 480 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys AlaLeu Phe Val 145 150 155 160 ctg gtg gct ccc agc tgc gcc tac cag gtg tgcggg ccg ccg ctg tac 528 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys GlyPro Pro Leu Tyr 165 170 175 cag ctc ggc gct gcc act cag gcc cgg ccc ccgcca cac gct agt gga 576 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro ProHis Ala Ser Gly 180 185 190 ccc cga agg cgt ctg gga tgc gaa cgg gcc tggaac cat agc gtc agg 624 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp AsnHis Ser Val Arg 195 200 205 gag gcc ggg gtc ccc ctg ggc ctg cca gcc ccgggt gcg agg agg cgc 672 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro GlyAla Arg Arg Arg 210 215 220 ggg ggc agt gcc agc cga agt ctg ccg ttg cccaag agg ccc agg cgt 720 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro LysArg Pro Arg Arg 225 230 235 240 ggc gct gcc cct gag ccg gag cgg acg cccgtt ggg cag ggg tcc tgg 768 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro ValGly Gln Gly Ser Trp 245 250 255 gcc cac ccg ggc agg acg cgt gga ccg agtgac cgt ggt ttc tgt gtg 816 Ala His Pro Gly Arg Thr Arg Gly Pro Ser AspArg Gly Phe Cys Val 260 265 270 gtg tca cct gcc aga ccc gcc gaa gaa gccacc tct ttg gag ggt gcg 864 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala ThrSer Leu Glu Gly Ala 275 280 285 ctc tct ggc acg cgc cac tcc cac cca tccgtg ggc cgc cag cac cac 912 Leu Ser Gly Thr Arg His Ser His Pro Ser ValGly Arg Gln His His 290 295 300 gcg ggc ccc cca tcc aca tcg cgg cca ccacgt ccc tgg gac acg cct 960 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro ArgPro Trp Asp Thr Pro 305 310 315 320 tgt ccc ccg gtg tac gcc gag acc aagcac ttc ctc tac tcc tca ggc 1008 Cys Pro Pro Val Tyr Ala Glu Thr Lys HisPhe Leu Tyr Ser Ser Gly 325 330 335 gac aag gag cag ctg cgg ccc tcc ttccta ctc agc tct ctg agg ccc 1056 Asp Lys Glu Gln Leu Arg Pro Ser Phe LeuLeu Ser Ser Leu Arg Pro 340 345 350 agc ctg act ggc gct cgg agg ctc gtggag acc atc ttt ctg ggt tcc 1104 Ser Leu Thr Gly Ala Arg Arg Leu Val GluThr Ile Phe Leu Gly Ser 355 360 365 agg ccc tgg atg cca ggg act ccc cgcagg ttg ccc cgc ctg ccc cag 1152 Arg Pro Trp Met Pro Gly Thr Pro Arg ArgLeu Pro Arg Leu Pro Gln 370 375 380 cgc tac tgg caa atg cgg ccc ctg tttctg gag ctg ctt ggg aac cac 1200 Arg Tyr Trp Gln Met Arg Pro Leu Phe LeuGlu Leu Leu Gly Asn His 385 390 395 400 gcg cag tgc ccc tac ggg gtg ctcctc aag acg cac tgc ccg ctg cga 1248 Ala Gln Cys Pro Tyr Gly Val Leu LeuLys Thr His Cys Pro Leu Arg 405 410 415 gct gcg gtc acc cca gca gcc ggtgtc tgt gcc cgg gag aag ccc cag 1296 Ala Ala Val Thr Pro Ala Ala Gly ValCys Ala Arg Glu Lys Pro Gln 420 425 430 ggc tct gtg gcg gcc ccc gag gaggag gac aca gac ccc cgt cgc ctg 1344 Gly Ser Val Ala Ala Pro Glu Glu GluAsp Thr Asp Pro Arg Arg Leu 435 440 445 gtg cag ctg ctc cgc cag cac agcagc ccc tgg cag gtg tac ggc ttc 1392 Val Gln Leu Leu Arg Gln His Ser SerPro Trp Gln Val Tyr Gly Phe 450 455 460 gtg cgg gcc tgc ctg cgc cgg ctggtg ccc cca ggc ctc tgg ggc tcc 1440 Val Arg Ala Cys Leu Arg Arg Leu ValPro Pro Gly Leu Trp Gly Ser 465 470 475 480 agg cac aac gaa cgc cgc ttcctc agg aac acc aag aag ttc atc tcc 1488 Arg His Asn Glu Arg Arg Phe LeuArg Asn Thr Lys Lys Phe Ile Ser 485 490 495 ctg ggg aag cat gcc aag ctctcg ctg cag gag ctg acg tgg aag atg 1536 Leu Gly Lys His Ala Lys Leu SerLeu Gln Glu Leu Thr Trp Lys Met 500 505 510 agc gtg cgg gac tgc gct tggctg cgc agg agc cca ggg gtt ggc tgt 1584 Ser Val Arg Asp Cys Ala Trp LeuArg Arg Ser Pro Gly Val Gly Cys 515 520 525 gtt ccg gcc gca gag cac cgtctg cgt gag gag atc ctg gcc aag ttc 1632 Val Pro Ala Ala Glu His Arg LeuArg Glu Glu Ile Leu Ala Lys Phe 530 535 540 ctg cac tgg ctg atg agt gtgtac gtc gtc gag ctg ctc agg tct ttc 1680 Leu His Trp Leu Met Ser Val TyrVal Val Glu Leu Leu Arg Ser Phe 545 550 555 560 ttt tat gtc acg gag accacg ttt caa aag aac agg ctc ttt ttc tac 1728 Phe Tyr Val Thr Glu Thr ThrPhe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 cgg aag agt gtc tgg agcaag ttg caa agc att gga atc aga cag cac 1776 Arg Lys Ser Val Trp Ser LysLeu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 ttg aag agg gtg cag ctgcgg gag ctg tcg gaa gca gag gtc agg cag 1824 Leu Lys Arg Val Gln Leu ArgGlu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 cat cgg gaa gcc agg cccgcc ctg ctg acg tcc aga ctc cgc ttc atc 1872 His Arg Glu Ala Arg Pro AlaLeu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 ccc aag cct gac ggg ctgcgg ccg att gtg aac atg gac tac gtc gtg 1920 Pro Lys Pro Asp Gly Leu ArgPro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 gga gcc aga acg ttccgc aga gaa aag agg gcc gag cgt ctc acc tcg 1968 Gly Ala Arg Thr Phe ArgArg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 agg gtg aag gca ctgttc agc gtg ctc aac tac gag cgg gcg cgg cgc 2016 Arg Val Lys Ala Leu PheSer Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 ccc ggc ctc ctg ggcgcc tct gtg ctg ggc ctg gac gat atc cac agg 2064 Pro Gly Leu Leu Gly AlaSer Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 gcc tgg cgc acc ttcgtg ctg cgt gtg cgg gcc cag gac ccg ccg cct 2112 Ala Trp Arg Thr Phe ValLeu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 gag ctg tac ttt gtcaag gtg gat gtg acg ggc gcg tac gac acc atc 2160 Glu Leu Tyr Phe Val LysVal Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 ccc cag gac aggctc acg gag gtc atc gcc agc atc atc aaa ccc cag 2208 Pro Gln Asp Arg LeuThr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 aac acg tac tgcgtg cgt cgg tat gcc gtg gtc cag aag gcc gcc cat 2256 Asn Thr Tyr Cys ValArg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 ggg cac gtc cgcaag gcc ttc aag agc cac gtc tct acc ttg aca gac 2304 Gly His Val Arg LysAla Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 ctc cag ccg tacatg cga cag ttc gtg gct cac ctg cag gag acc agc 2352 Leu Gln Pro Tyr MetArg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 ccg ctg agg gatgcc gtc gtc atc gag cag agc tcc tcc ctg aat gag 2400 Pro Leu Arg Asp AlaVal Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 gcc agc agtggc ctc ttc gac gtc ttc cta cgc ttc atg tgc cac cac 2448 Ala Ser Ser GlyLeu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 gcc gtg cgcatc agg ggc aag tcc tac gtc cag tgc cag ggg atc ccg 2496 Ala Val Arg IleArg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 cag ggc tccatc ctc tcc acg ctg ctc tgc agc ctg tgc tac ggc gac 2544 Gln Gly Ser IleLeu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 atg gag aacaag ctg ttt gcg ggg att cgg cgg gac ggg ctg ctc ctg 2592 Met Glu Asn LysLeu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 cgt ttg gtggat gat ttc ttg ttg gtg aca cct cac ctc acc cac gcg 2640 Arg Leu Val AspAsp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 aaa accttc ctc agg acc ctg gtc cga ggt gtc cct gag tat ggc tgc 2688 Lys Thr PheLeu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 gtg gtgaac ttg cgg aag aca gtg gtg aac ttc cct gta gaa gac gag 2736 Val Val AsnLeu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 gcc ctgggt ggc acg gct ttt gtt cag atg ccg gcc cac ggc cta ttc 2784 Ala Leu GlyGly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 ccc tggtgc ggc ctg ctg ctg gat acc cgg acc ctg gag gtg cag agc 2832 Pro Trp CysGly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 gac tactcc agc tat gcc cgg acc tcc atc aga gcc agt ctc acc ttc 2880 Asp Tyr SerSer Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 aaccgc ggc ttc aag gct ggg agg aac atg cgt cgc aaa ctc ttt ggg 2928 Asn ArgGly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 gtcttg cgg ctg aag tgt cac agc ctg ttt ctg gat ttg cag gtg aac 2976 Val LeuArg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 agcctc cag acg gtg tgc acc aac atc tac aag atc ctc ctg ctg cag 3024 Ser LeuGln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005gcg tac agg ttt cac gca tgt gtg ctg cag ctc cca ttt cat cag caa 3072 AlaTyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 10151020 gtt tgg aag aac ccc aca ttt ttc ctg cgc gtc atc tct gac acg gcc3120 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala1025 1030 1035 1040 tcc ctc tgc tac tcc atc ctg aaa gcc aag aac gca gggatg tcg ctg 3168 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly MetSer Leu 1045 1050 1055 ggg gcc aag ggc gcc gcc ggc cct ctg ccc tcc gaggcc gtg cag tgg 3216 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu AlaVal Gln Trp 1060 1065 1070 ctg tgc cac caa gca ttc ctg ctc aag ctg actcga cac cgt gtc acc 3264 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr ArgHis Arg Val Thr 1075 1080 1085 tac gtg cca ctc ctg ggg tca ctc agg acagcc cag acg cag ctg agt 3312 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr AlaGln Thr Gln Leu Ser 1090 1095 1100 cgg aag ctc ccg ggg acg acg ctg actgcc ctg gag gcc gca gcc aac 3360 Arg Lys Leu Pro Gly Thr Thr Leu Thr AlaLeu Glu Ala Ala Ala Asn 1105 1110 1115 1120 ccg gca ctg ccc tca gac ttcaag acc atc ctg gac 3396 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp1125 1130 33 21 DNA Artificial Sequence Description of ArtificialSequence artificially synthesized primer sequence 33 ttggcttccaggccataatt g 21 34 20 DNA Artificial Sequence Description of ArtificialSequence artificially synthesized primer sequence 34 aagagggcagatctatcgga 20 35 20 DNA Artificial Sequence Description of ArtificialSequence artificially synthesized primer sequence 35 atggatctcctgaaggtgct 20 36 20 DNA Artificial Sequence Description of ArtificialSequence artificially synthesized primer sequence 36 aagagggcagatctatcgga 20 37 23 DNA Artificial Sequence Description of ArtificialSequence artificially synthesized primer sequence 37 ggaagagtgagcggccatca agg 23 38 22 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 38ctgctggaga ggttattcct cg 22 39 24 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 39gccaacacca acctgtccaa gttc 24 40 24 DNA Artificial Sequence Descriptionof Artificial Sequence artificially synthesized primer sequence 40tgcaaaggct ccaggtctga gggc 24 41 19 DNA Artificial Sequence Descriptionof Artificial Sequence artificially synthesized primer sequence 41ctctctctcc tcaggacaa 19 42 22 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 42tggagcaaaa cagaatggct gg 22 43 24 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 43ctgagatgtc tctctctctc ttag 24 44 20 DNA Artificial Sequence Descriptionof Artificial Sequence artificially synthesized primer sequence 44acaatgactg atgagagatg 20 45 18 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 45cagacctgaa ggagacct 18 46 18 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 46gtcagcgtaa acagttgc 18 47 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 47gccaagaagc ggatagaagg 20 48 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 48ctgtggttca gggctcagtc 20 49 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 49cagtggagct ggacaaagcc 20 50 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 50tagcgacggt tctggaacca 20 51 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 51ctgtcatctc actatgggca 20 52 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 52ccaagtccga gcaggaattt 20 53 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 53aagacgtcaa gccctttgtg 20 54 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 54aaaggagcac actttggtgg 20 55 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 55agcaagaata cgatgccatc 20 56 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 56gaaggggtgg tggtacggtc 20 57 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 57tgggaatggc tatgtcagtg 20 58 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 58ctggtaatct gtgttgtagg 20 59 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 59caagggcctc tccaaacttg 20 60 20 DNA Artificial Sequence Description ofArtificial Sequence artificially synthesized primer sequence 60gccccagaga cagcattcca 20 61 268 PRT Homo sapiens 61 Met Ala Gln Pro LeuCys Pro Pro Leu Ser Glu Ser Trp Met Leu Ser 1 5 10 15 Ala Ala Trp GlyPro Thr Arg Arg Pro Pro Pro Ser Asp Lys Asp Cys 20 25 30 Gly Arg Ser LeuVal Ser Ser Pro Asp Ser Trp Gly Ser Thr Pro Ala 35 40 45 Asp Ser Pro ValAla Ser Pro Ala Arg Pro Gly Thr Leu Arg Asp Pro 50 55 60 Arg Ala Pro SerVal Gly Arg Arg Gly Ala Arg Ser Ser Arg Leu Gly 65 70 75 80 Ser Gly GlnArg Gln Ser Ala Ser Glu Arg Glu Lys Leu Arg Met Arg 85 90 95 Thr Leu AlaArg Ala Leu His Glu Leu Arg Arg Phe Leu Pro Pro Ser 100 105 110 Val AlaPro Ala Gly Gln Ser Leu Thr Lys Ile Glu Thr Leu Arg Leu 115 120 125 AlaIle Arg Tyr Ile Gly His Leu Ser Ala Val Leu Gly Leu Ser Glu 130 135 140Glu Ser Leu Gln Arg Arg Cys Arg Gln Arg Gly Asp Ala Gly Ser Pro 145 150155 160 Arg Gly Cys Pro Leu Cys Pro Asp Asp Cys Pro Ala Gln Met Gln Thr165 170 175 Arg Thr Gln Ala Glu Gly Gln Gly Gln Gly Arg Gly Leu Gly LeuVal 180 185 190 Ser Ala Val Arg Ala Gly Ala Ser Trp Gly Ser Pro Pro AlaCys Pro 195 200 205 Gly Ala Arg Ala Ala Pro Glu Pro Arg Asp Pro Pro AlaLeu Phe Ala 210 215 220 Glu Ala Ala Cys Pro Glu Gly Gln Ala Met Glu ProSer Pro Pro Ser 225 230 235 240 Pro Leu Leu Pro Gly Asp Val Leu Ala LeuLeu Glu Thr Trp Met Pro 245 250 255 Leu Ser Pro Leu Glu Trp Leu Pro GluGlu Pro Lys 260 265 62 804 DNA Homo sapiens CDS (1)..(807) 62 atg gcccag ccc ctg tgc ccg ccg ctc tcc gag tcc tgg atg ctc tct 48 Met Ala GlnPro Leu Cys Pro Pro Leu Ser Glu Ser Trp Met Leu Ser 1 5 10 15 gcg gcctgg ggc cca act cgg cgg ccg ccg ccc tcc gac aag gac tgc 96 Ala Ala TrpGly Pro Thr Arg Arg Pro Pro Pro Ser Asp Lys Asp Cys 20 25 30 ggc cgc tccctc gtc tcg tcc cca gac tca tgg ggc agc acc cca gcc 144 Gly Arg Ser LeuVal Ser Ser Pro Asp Ser Trp Gly Ser Thr Pro Ala 35 40 45 gac agc ccc gtggcg agc ccc gcg cgg cca ggc acc ctc cgg gac ccc 192 Asp Ser Pro Val AlaSer Pro Ala Arg Pro Gly Thr Leu Arg Asp Pro 50 55 60 cgc gcc ccc tcc gtaggt agg cgc ggc gcg cgc agc agc cgc ctg ggc 240 Arg Ala Pro Ser Val GlyArg Arg Gly Ala Arg Ser Ser Arg Leu Gly 65 70 75 80 agc ggg cag agg cagagc gcc agt gag cgg gag aaa ctg cgc atg cgc 288 Ser Gly Gln Arg Gln SerAla Ser Glu Arg Glu Lys Leu Arg Met Arg 85 90 95 acg ctg gcc cgc gcc ctgcac gag ctg cgc cgc ttt cta ccg ccg tcc 336 Thr Leu Ala Arg Ala Leu HisGlu Leu Arg Arg Phe Leu Pro Pro Ser 100 105 110 gtg gcg ccc gcg ggc cagagc ctg acc aag atc gag acg ctg cgc ctg 384 Val Ala Pro Ala Gly Gln SerLeu Thr Lys Ile Glu Thr Leu Arg Leu 115 120 125 gct atc cgc tat atc ggccac ctg tcg gcc gtg cta ggc ctc agc gag 432 Ala Ile Arg Tyr Ile Gly HisLeu Ser Ala Val Leu Gly Leu Ser Glu 130 135 140 gag agt ctc cag cgc cggtgc cgg cag cgc ggt gac gcg ggg tcc cct 480 Glu Ser Leu Gln Arg Arg CysArg Gln Arg Gly Asp Ala Gly Ser Pro 145 150 155 160 cgg ggc tgc ccg ctgtgc ccc gac gac tgc ccc gcg cag atg cag aca 528 Arg Gly Cys Pro Leu CysPro Asp Asp Cys Pro Ala Gln Met Gln Thr 165 170 175 cgg acg cag gct gagggg cag ggg cag ggg cgc ggg ctg ggc ctg gta 576 Arg Thr Gln Ala Glu GlyGln Gly Gln Gly Arg Gly Leu Gly Leu Val 180 185 190 tcc gcc gtc cgc gccggg gcg tcc tgg gga tcc ccg cct gcc tgc ccc 624 Ser Ala Val Arg Ala GlyAla Ser Trp Gly Ser Pro Pro Ala Cys Pro 195 200 205 gga gcc cga gct gcaccc gag ccg cgc gac ccg cct gcg ctg ttc gcc 672 Gly Ala Arg Ala Ala ProGlu Pro Arg Asp Pro Pro Ala Leu Phe Ala 210 215 220 gag gcg gcg tgc cctgaa ggg cag gcg atg gag cca agc cca ccg tcc 720 Glu Ala Ala Cys Pro GluGly Gln Ala Met Glu Pro Ser Pro Pro Ser 225 230 235 240 ccg ctc ctt ccgggc gac gtg ctg gct ctg ttg gag acc tgg atg ccc 768 Pro Leu Leu Pro GlyAsp Val Leu Ala Leu Leu Glu Thr Trp Met Pro 245 250 255 ctc tcg cct ctggag tgg ctg cct gag gag ccc aag 804 Leu Ser Pro Leu Glu Trp Leu Pro GluGlu Pro Lys 260 265 63 215 PRT Homo sapiens 63 Met Gly Ser Pro Arg SerAla Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu GlnAla Gln Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30 Thr Gln His Val ArgGlu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35 40 45 Arg Leu Ile Arg ThrTyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60 Val Gln Val Leu AlaAsn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly 65 70 75 80 Asp Pro Phe AlaLys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg 85 90 95 Val Arg Val ArgGly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys 100 105 110 Lys Gly LysLeu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val 115 120 125 Phe ThrGlu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala 130 135 140 LysTyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155160 Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His Phe Met Lys 165170 175 Arg Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu180 185 190 Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser GlnArg 195 200 205 Thr Trp Ala Pro Glu Pro Arg 210 215 64 645 DNA Homosapiens CDS (1)..(648) 64 atg ggc agc ccc cgc tcc gcg ctg agc tgc ctgctg ttg cac ttg ctg 48 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu LeuLeu His Leu Leu 1 5 10 15 gtc ctc tgc ctc caa gcc cag gta act gtt cagtcc tca cct aat ttt 96 Val Leu Cys Leu Gln Ala Gln Val Thr Val Gln SerSer Pro Asn Phe 20 25 30 aca cag cat gtg agg gag cag agc ctg gtg acg gatcag ctc agc cgc 144 Thr Gln His Val Arg Glu Gln Ser Leu Val Thr Asp GlnLeu Ser Arg 35 40 45 cgc ctc atc cgg acc tac caa ctc tac agc cgc acc agcggg aag cac 192 Arg Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser GlyLys His 50 55 60 gtg cag gtc ctg gcc aac aag cgc atc aac gcc atg gca gaggac ggc 240 Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu AspGly 65 70 75 80 gac ccc ttc gca aag ctc atc gtg gag acg gac acc ttt ggaagc aga 288 Asp Pro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly SerArg 85 90 95 gtt cga gtc cga gga gcc gag acg ggc ctc tac atc tgc atg aacaag 336 Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys100 105 110 aag ggg aag ctg atc gcc aag agc aac ggc aaa ggc aag gac tgcgtc 384 Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val115 120 125 ttc acg gag att gtg ctg gag aac aac tac aca gcg ctg cag aatgcc 432 Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala130 135 140 aag tac gag ggc tgg tac atg gcc ttc acc cgc aag ggc cgg ccccgc 480 Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg145 150 155 160 aag ggc tcc aag acg cgg cag cac cag cgt gag gtc cac ttcatg aag 528 Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His Phe MetLys 165 170 175 cgg ctg ccc cgg ggc cac cac acc acc gag cag agc ctg cgcttc gag 576 Arg Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg PheGlu 180 185 190 ttc ctc aac tac ccg ccc ttc acg cgc agc ctg cgc ggc agccag agg 624 Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser GlnArg 195 200 205 act tgg gcc ccg gaa ccc cga 645 Thr Trp Ala Pro Glu ProArg 210 215 65 212 PRT Homo sapiens 65 Met Asp Tyr Leu Leu Met Ile PheSer Leu Leu Phe Val Ala Cys Gln 1 5 10 15 Gly Ala Pro Glu Thr Ala ValLeu Gly Ala Glu Leu Ser Ala Val Gly 20 25 30 Glu Asn Gly Gly Glu Lys ProThr Pro Ser Pro Pro Trp Arg Leu Arg 35 40 45 Arg Ser Lys Arg Cys Ser CysSer Ser Leu Met Asp Lys Glu Cys Val 50 55 60 Tyr Phe Cys His Leu Asp IleIle Trp Val Asn Thr Pro Glu His Val 65 70 75 80 Val Pro Tyr Gly Leu GlySer Pro Arg Ser Lys Arg Ala Leu Glu Asn 85 90 95 Leu Leu Pro Thr Lys AlaThr Asp Arg Glu Asn Arg Cys Gln Cys Ala 100 105 110 Ser Gln Lys Asp LysLys Cys Trp Asn Phe Cys Gln Ala Gly Lys Glu 115 120 125 Leu Arg Ala GluAsp Ile Met Glu Lys Asp Trp Asn Asn His Lys Lys 130 135 140 Gly Lys AspCys Ser Lys Leu Gly Lys Lys Cys Ile Tyr Gln Gln Leu 145 150 155 160 ValArg Gly Arg Lys Ile Arg Arg Ser Ser Glu Glu His Leu Arg Gln 165 170 175Thr Arg Ser Glu Thr Met Arg Asn Ser Val Lys Ser Ser Phe His Asp 180 185190 Pro Lys Leu Lys Gly Lys Pro Ser Arg Glu Arg Tyr Val Thr His Asn 195200 205 Arg Ala His Trp 210 66 636 DNA Homo sapiens CDS (1)..(639) 66atg gat tat ttg ctc atg att ttc tct ctg ctg ttt gtg gct tgc caa 48 MetAsp Tyr Leu Leu Met Ile Phe Ser Leu Leu Phe Val Ala Cys Gln 1 5 10 15gga gct cca gaa aca gca gtc tta ggc gct gag ctc agc gcg gtg ggt 96 GlyAla Pro Glu Thr Ala Val Leu Gly Ala Glu Leu Ser Ala Val Gly 20 25 30 gagaac ggc ggg gag aaa ccc act ccc agt cca ccc tgg cgg ctc cgc 144 Glu AsnGly Gly Glu Lys Pro Thr Pro Ser Pro Pro Trp Arg Leu Arg 35 40 45 cgg tccaag cgc tgc tcc tgc tcg tcc ctg atg gat aaa gag tgt gtc 192 Arg Ser LysArg Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val 50 55 60 tac ttc tgccac ctg gac atc att tgg gtc aac act ccc gag cac gtt 240 Tyr Phe Cys HisLeu Asp Ile Ile Trp Val Asn Thr Pro Glu His Val 65 70 75 80 gtt ccg tatgga ctt gga agc cct agg tcc aag aga gcc ttg gag aat 288 Val Pro Tyr GlyLeu Gly Ser Pro Arg Ser Lys Arg Ala Leu Glu Asn 85 90 95 tta ctt ccc acaaag gca aca gac cgt gag aat aga tgc caa tgt gct 336 Leu Leu Pro Thr LysAla Thr Asp Arg Glu Asn Arg Cys Gln Cys Ala 100 105 110 agc caa aaa gacaag aag tgc tgg aat ttt tgc caa gca gga aaa gaa 384 Ser Gln Lys Asp LysLys Cys Trp Asn Phe Cys Gln Ala Gly Lys Glu 115 120 125 ctc agg gct gaagac att atg gag aaa gac tgg aat aat cat aag aaa 432 Leu Arg Ala Glu AspIle Met Glu Lys Asp Trp Asn Asn His Lys Lys 130 135 140 gga aaa gac tgttcc aag ctt ggg aaa aag tgt att tat cag cag tta 480 Gly Lys Asp Cys SerLys Leu Gly Lys Lys Cys Ile Tyr Gln Gln Leu 145 150 155 160 gtg aga ggaaga aaa atc aga aga agt tca gag gaa cac cta aga caa 528 Val Arg Gly ArgLys Ile Arg Arg Ser Ser Glu Glu His Leu Arg Gln 165 170 175 acc agg tcggag acc atg aga aac agc gtc aaa tca tct ttt cat gat 576 Thr Arg Ser GluThr Met Arg Asn Ser Val Lys Ser Ser Phe His Asp 180 185 190 ccc aag ctgaaa ggc aag ccc tcc aga gag cgt tat gtg acc cac aac 624 Pro Lys Leu LysGly Lys Pro Ser Arg Glu Arg Tyr Val Thr His Asn 195 200 205 cga gca cattgg 636 Arg Ala His Trp 210 67 143 PRT Homo sapiens 67 Met Gln His ArgGly Phe Leu Leu Leu Thr Leu Leu Ala Leu Leu Ala 1 5 10 15 Leu Thr SerAla Val Ala Lys Lys Lys Asp Lys Val Lys Lys Gly Gly 20 25 30 Pro Gly SerGlu Cys Ala Glu Trp Ala Trp Gly Pro Cys Thr Pro Ser 35 40 45 Ser Lys AspCys Gly Val Gly Phe Arg Glu Gly Thr Cys Gly Ala Gln 50 55 60 Thr Gln ArgIle Arg Cys Arg Val Pro Cys Asn Trp Lys Lys Glu Phe 65 70 75 80 Gly AlaAsp Cys Lys Tyr Lys Phe Glu Asn Trp Gly Ala Cys Asp Gly 85 90 95 Gly ThrGly Thr Lys Val Arg Gln Gly Thr Leu Lys Lys Ala Arg Tyr 100 105 110 AsnAla Gln Cys Gln Glu Thr Ile Arg Val Thr Lys Pro Cys Thr Pro 115 120 125Lys Thr Lys Ala Lys Ala Lys Ala Lys Lys Gly Lys Gly Lys Asp 130 135 14068 429 DNA Homo sapiens CDS (1)..(432) 68 atg cag cac cga ggc ttc ctcctc ctc acc ctc ctc gcc ctg ctg gcg 48 Met Gln His Arg Gly Phe Leu LeuLeu Thr Leu Leu Ala Leu Leu Ala 1 5 10 15 ctc acc tcc gcg gtc gcc aaaaag aaa gat aag gtg aag aag ggc ggc 96 Leu Thr Ser Ala Val Ala Lys LysLys Asp Lys Val Lys Lys Gly Gly 20 25 30 ccg ggg agc gag tgc gct gag tgggcc tgg ggg ccc tgc acc ccc agc 144 Pro Gly Ser Glu Cys Ala Glu Trp AlaTrp Gly Pro Cys Thr Pro Ser 35 40 45 agc aag gat tgc ggc gtg ggt ttc cgcgag ggc acc tgc ggg gcc cag 192 Ser Lys Asp Cys Gly Val Gly Phe Arg GluGly Thr Cys Gly Ala Gln 50 55 60 acc cag cgc atc cgg tgc agg gtg ccc tgcaac tgg aag aag gag ttt 240 Thr Gln Arg Ile Arg Cys Arg Val Pro Cys AsnTrp Lys Lys Glu Phe 65 70 75 80 gga gcc gac tgc aag tac aag ttt gag aactgg ggt gcg tgt gat ggg 288 Gly Ala Asp Cys Lys Tyr Lys Phe Glu Asn TrpGly Ala Cys Asp Gly 85 90 95 ggc aca ggc acc aaa gtc cgc caa ggc acc ctgaag aag gcg cgc tac 336 Gly Thr Gly Thr Lys Val Arg Gln Gly Thr Leu LysLys Ala Arg Tyr 100 105 110 aat gct cag tgc cag gag acc atc cgc gtc accaag ccc tgc acc ccc 384 Asn Ala Gln Cys Gln Glu Thr Ile Arg Val Thr LysPro Cys Thr Pro 115 120 125 aag acc aaa gca aag gcc aaa gcc aag aaa gggaag gga aag gac 429 Lys Thr Lys Ala Lys Ala Lys Ala Lys Lys Gly Lys GlyLys Asp 130 135 140 69 408 PRT Homo sapiens 69 Met Ile Pro Gly Asn ArgMet Leu Met Val Val Leu Leu Cys Gln Val 1 5 10 15 Leu Leu Gly Gly AlaSer His Ala Ser Leu Ile Pro Glu Thr Gly Lys 20 25 30 Lys Lys Val Ala GluIle Gln Gly His Ala Gly Gly Arg Arg Ser Gly 35 40 45 Gln Ser His Glu LeuLeu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met 50 55 60 Phe Gly Leu Arg ArgArg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro 65 70 75 80 Asp Tyr Met ArgAsp Leu Tyr Arg Leu Gln Ser Gly Glu Glu Glu Glu 85 90 95 Glu Gln Ile HisSer Thr Gly Leu Glu Tyr Pro Glu Arg Pro Ala Ser 100 105 110 Arg Ala AsnThr Val Arg Ser Phe His His Glu Glu His Leu Glu Asn 115 120 125 Ile ProGly Thr Ser Glu Asn Ser Ala Phe Arg Phe Leu Phe Asn Leu 130 135 140 SerSer Ile Pro Glu Asn Glu Ala Ile Ser Ser Ala Glu Leu Arg Leu 145 150 155160 Phe Arg Glu Gln Val Asp Gln Gly Pro Asp Trp Glu Arg Gly Phe His 165170 175 Arg Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala Glu Val Val Pro180 185 190 Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg Leu Val His HisAsn 195 200 205 Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pro Ala Val LeuArg Trp 210 215 220 Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Ala Ile GluVal Thr His 225 230 235 240 Leu His Gln Thr Arg Thr His Gln Gly Gln HisVal Arg Ile Ser Arg 245 250 255 Ser Leu Pro Gln Gly Ser Gly Asn Trp AlaGln Leu Arg Pro Leu Leu 260 265 270 Val Thr Phe Gly His Asp Gly Arg GlyHis Ala Leu Thr Arg Arg Arg 275 280 285 Arg Ala Lys Arg Ser Pro Lys HisHis Ser Gln Arg Ala Arg Lys Lys 290 295 300 Asn Lys Asn Cys Arg Arg HisSer Leu Tyr Val Asp Phe Ser Asp Val 305 310 315 320 Gly Trp Asn Asp TrpIle Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr 325 330 335 Cys His Gly AspCys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr 340 345 350 Asn His AlaIle Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile 355 360 365 Pro LysAla Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu 370 375 380 TyrLeu Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met 385 390 395400 Val Val Glu Gly Cys Gly Cys Arg 405 70 1224 DNA Homo sapiens CDS(1)..(1227) 70 atg att cct ggt aac cga atg ctg atg gtc gtt tta tta tgccaa gtc 48 Met Ile Pro Gly Asn Arg Met Leu Met Val Val Leu Leu Cys GlnVal 1 5 10 15 ctg cta gga ggc gcg agc cat gct agt ttg ata cct gag acgggg aag 96 Leu Leu Gly Gly Ala Ser His Ala Ser Leu Ile Pro Glu Thr GlyLys 20 25 30 aaa aaa gtc gcc gag att cag ggc cac gcg gga gga cgc cgc tcaggg 144 Lys Lys Val Ala Glu Ile Gln Gly His Ala Gly Gly Arg Arg Ser Gly35 40 45 cag agc cat gag ctc ctg cgg gac ttc gag gcg aca ctt ctg cag atg192 Gln Ser His Glu Leu Leu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met 5055 60 ttt ggg ctg cgc cgc cgc ccg cag cct agc aag agt gcc gtc att ccg240 Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro 6570 75 80 gac tac atg cgg gat ctt tac cgg ctt cag tct ggg gag gag gag gaa288 Asp Tyr Met Arg Asp Leu Tyr Arg Leu Gln Ser Gly Glu Glu Glu Glu 8590 95 gag cag atc cac agc act ggt ctt gag tat cct gag cgc ccg gcc agc336 Glu Gln Ile His Ser Thr Gly Leu Glu Tyr Pro Glu Arg Pro Ala Ser 100105 110 cgg gcc aac acc gtg agg agc ttc cac cac gaa gaa cat ctg gag aac384 Arg Ala Asn Thr Val Arg Ser Phe His His Glu Glu His Leu Glu Asn 115120 125 atc cca ggg acc agt gaa aac tct gct ttt cgt ttc ctc ttt aac ctc432 Ile Pro Gly Thr Ser Glu Asn Ser Ala Phe Arg Phe Leu Phe Asn Leu 130135 140 agc agc atc cct gag aac gag gcg atc tcc tct gca gag ctt cgg ctc480 Ser Ser Ile Pro Glu Asn Glu Ala Ile Ser Ser Ala Glu Leu Arg Leu 145150 155 160 ttc cgg gag cag gtg gac cag ggc cct gat tgg gaa agg ggc ttccac 528 Phe Arg Glu Gln Val Asp Gln Gly Pro Asp Trp Glu Arg Gly Phe His165 170 175 cgt ata aac att tat gag gtt atg aag ccc cca gca gaa gtg gtgcct 576 Arg Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala Glu Val Val Pro180 185 190 ggg cac ctc atc aca cga cta ctg gac acg aga ctg gtc cac cacaat 624 Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg Leu Val His His Asn195 200 205 gtg aca cgg tgg gaa act ttt gat gtg agc cct gcg gtc ctt cgctgg 672 Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pro Ala Val Leu Arg Trp210 215 220 acc cgg gag aag cag cca aac tat ggg cta gcc att gag gtg actcac 720 Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Ala Ile Glu Val Thr His225 230 235 240 ctc cat cag act cgg acc cac cag ggc cag cat gtc agg attagc cga 768 Leu His Gln Thr Arg Thr His Gln Gly Gln His Val Arg Ile SerArg 245 250 255 tcg tta cct caa ggg agt ggg aat tgg gcc cag ctc cgg cccctc ctg 816 Ser Leu Pro Gln Gly Ser Gly Asn Trp Ala Gln Leu Arg Pro LeuLeu 260 265 270 gtc acc ttt ggc cat gat ggc cgg ggc cat gcc ttg acc cgacgc cgg 864 Val Thr Phe Gly His Asp Gly Arg Gly His Ala Leu Thr Arg ArgArg 275 280 285 agg gcc aag cgt agc cct aag cat cac tca cag cgg gcc aggaag aag 912 Arg Ala Lys Arg Ser Pro Lys His His Ser Gln Arg Ala Arg LysLys 290 295 300 aat aag aac tgc cgg cgc cac tcg ctc tat gtg gac ttc agcgat gtg 960 Asn Lys Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser AspVal 305 310 315 320 ggc tgg aat gac tgg att gtg gcc cca cca ggc tac caggcc ttc tac 1008 Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln AlaPhe Tyr 325 330 335 tgc cat ggg gac tgc ccc ttt cca ctg gct gac cac ctcaac tca acc 1056 Cys His Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu AsnSer Thr 340 345 350 aac cat gcc att gtg cag acc ctg gtc aat tct gtc aattcc agt atc 1104 Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn SerSer Ile 355 360 365 ccc aaa gcc tgt tgt gtg ccc act gaa ctg agt gcc atctcc atg ctg 1152 Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile SerMet Leu 370 375 380 tac ctg gat gag tat gat aag gtg gta ctg aaa aat tatcag gag atg 1200 Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr GlnGlu Met 385 390 395 400 gta gta gag gga tgt ggg tgc cgc 1224 Val Val GluGly Cys Gly Cys Arg 405 71 24 DNA Artificial Sequence 71 gcccgcgctccaactgctct gatg 24 72 24 DNA Artificial Sequence 72 tgcctacggtggtgcgccct ctgc 24 73 22 DNA Artificial Sequence 73 gaagcgcaacagggccatca cg 22 74 22 DNA Artificial Sequence 74 ccacgtcacg caggtcccgttc 22 75 22 DNA Artificial Sequence 75 gatcctgttc tctgcctctg ga 22 76 22DNA Artificial Sequence 76 tcatccactt tgtccacccg ag 22 77 21 DNAArtificial Sequence 77 ttcctcgtct tggccttttg g 21 78 21 DNA ArtificialSequence 78 gctggatctt cgtaggctcc g 21 79 19 DNA Artificial Sequence 79ggcaagctga ccctgaagt 19 80 19 DNA Artificial Sequence 80 gggtgctcaggtagtggtt 19

1. A cell which has been isolated from a living tissue or umbilicalblood, and which has the potential to differentiate into at least acardiomyocyte.
 2. The cell according to claim 1, wherein the livingtissue is bone marrow.
 3. The cell according to claim 1 or 2, whereinthe cell is a multipotential stem cell.
 4. The cell according to any oneof claims 1 to 3, wherein the cell is a multipotential stem cell whichdifferentiates into at least a cardiomyocyte and a vascular endothelialcell.
 5. The cell according to any one of claims 1 to 4, wherein thecell is a multipotential stem cell which differentiates into at least acardiomyocyte, an adipocyte, a skeletal muscle cell, an osteoblast, anda vascular endothelial cell.
 6. The cell according to any one of claims1 to 5, wherein the cell is a multipotential stem cell whichdifferentiates into at least a cardiomyocyte, an adipocyte, a skeletalmuscle cell, an osteoblast, a vascular endothelial cell, a nervous cell,and a hepatic cell.
 7. The cell according to any one of claims 1 to 3,wherein the cell is a multipotential stem cell which differentiates intoany cell in adult tissues.
 8. The cell according to any one of claims 1to 7, wherein the cell is CD117-positive and CD140-positive.
 9. The cellaccording to claim 8, wherein the cell is further CD34-positive.
 10. Thecell according to claim 9, wherein the cell is further CD144-positive.11. The cell according to claim 9, wherein the cell is furtherCD140-negative.
 12. The cell according to claim 8, wherein the cell isCD34-negative.
 13. The cell according to claim 12, wherein the cell isfurther CD144-positive.
 14. The cell according to claim 12, wherein thecell is further CD144-negative.
 15. The cell according to claim 10,wherein the cell is further CD14-negative, CD45-negative, CD90-negative,Flk-1-negative, CD31-negative, CD105-negative, CD49b-negative,CD49d-negative, CD29-positive, CD54-negative, CD102-negative,CD106-negative, and CD44-positive.
 16. The cell according to claim 11,wherein the cell is further CD14-negative, CD45-negative, CD90-negative,Flk-1-negative, CD31-negative, CD105-negative, CD49b-negative,CD49d-negative, CD29-positive, CD54-negative, CD102-negative,CD106-negative, and CD44-positive.
 17. The cell according to claim 12,wherein the cell is further CD14-negative, CD45-negative, CD90-negative,Flk-l-negative, CD31-negative, CD105-negative, CD49b-negative,CD49d-negative, CD29-positive, CD54-negative, CD102-negative,CD106-negative, and CD44-positive.
 18. The cell according to claim 13,wherein the cell is further CD14-negative, CD45-negative, CD90-negative,Flk-1-negative, CD31-negative, CD105-negative, CD49b-negative,CD49d-negative, CD29-positive, CD54-negative, CD102-negative,CD106-negative, and CD44-positive.
 19. The cell according to claim 1,which does not take up Hoechst
 33342. 20. A cardiomyocyte precursorwhich differentiates into only cardiomyocyte induced from the cellaccording to any one of claims 1 to
 19. 21. The cell according to anyone of claims 1 to 20, which has the potential to differentiate into aventricular cardiac muscle cell.
 22. The cell according to any one ofclaims 1 to 20, which has the potential to differentiate into a sinusnode cell.
 23. The cell according to any one of claims 1 to 20, whereinthe vital tissue or umbilical blood is derived from a mammal.
 24. Thecell according to claim 23, wherein the mammal is selected from thegroup consisting of a mouse, a rat, a guinea pig, a hamster, a rabbit, acat, a dog, a sheep, a swine, cattle, a goat and a human.
 25. The cellaccording to any one of claims 1 to 8, which is mouse bonemarrow-derived multipotential stem cell BMSC (FERM BP-7043).
 26. Thecell according to any one of claims 1 to 25, which has the potential todifferentiate into a cardiomyocyte by demethylation of a chromosomal DNAof the cell.
 27. The cell according to claim 26, wherein thedemethylation is carried out by at least one selected from the groupconsisting of demethylase, 5-azacytidine, and dimethyl sulfoxide, DMSO.28. The cell according to claim 27, wherein the demethylase comprisesthe amino acid sequence represented by SEQ ID NO:1.
 29. The cellaccording to any one of claims 1 to 28, wherein the differentiation isaccelerated by a factor which is expressed in a cardiogenesis region ofa fetus or a factor which acts on differentiation into a cardiomyocytein a cardiogenesis stage of a fetus.
 30. The cell according to claim 29,wherein the factor which is expressed in a cardiogenesis region of afetus or the factor which acts on differentiation into a cardiomyocytein a cardiogenesis stage of a fetus is at least one selected from thegroup consisting of a cytokine, an adhesion molecule, a vitamin, atranscription factor, and an extracellular matrix.
 31. The cellaccording to claim 30, wherein the cytokine is at least one selectedfrom the group consisting of a platelet-derived growth factor, PDGF; afibroblast growth factor-8, FGF-8; an endothelin 1, ET1; a midkine; anda bone morphogenetic factor, BMP-4.
 32. The cell according to claim 31,wherein the PDGF, FGF-8, ET1, midkine, and BMP-4 comprise the amino acidsequence represented by SEQ ID NO:3 or 5, the amino acid sequencerepresented by SEQ ID NO:64, the amino acid sequence represented by SEQID NO:66, the amino acid sequence represented by SEQ ID NO:68, and theamino acid sequence represented by SEQ ID NO:70, respectively.
 33. Thecell according to claim 30, wherein the adhesion molecule is at leastone selected from the group consisting of a gelatin, a laminin, acollagen, and a fibronectin.
 34. The cell according to claim 30, whereinthe vitamin is retinoic acid.
 35. The cell according to claim 30,wherein the transcription factor is at least one selected from the groupconsisting of Nkx2.5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND,eHAND, TEF-1, TEF-3, TEF-5, and MesP1.
 36. The cell according to claim35, wherein the Nkx2.5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D,dHAND, eHAND, TEF-1, TEF-3, TEF-5, and MesP1 comprise the amino acidsequence represented by SEQ ID NO:9, the amino acid sequence representedby SEQ ID NO:11, the amino acid sequence represented by SEQ ID NO:13,the amino acid sequence represented by SEQ ID NO:15, the amino acidsequence represented by SEQ ID NO:17, the amino acid sequencerepresented by SEQ ID NO:19, the amino acid sequence represented by SEQID NO:21, the amino acid sequence represented by SEQ ID NO:23, the aminoacid sequence represented by SEQ ID NO:25, the amino acid sequencerepresented by SEQ ID NO:27, the amino acid sequence represented by SEQID NO:29, and the amino acid sequence represented by SEQ ID NO:62,respectively.
 37. The cell according to claim 30, wherein theextracellular matrix is an extracellular matrix derived from acardiomyocyte.
 38. The cell according to any one of claims 1 to 28,wherein the differentiation is inhibited by a fibroblast growthfactor-2, FGF-2.
 39. The cell according to claim 38, wherein the FGF-2comprises the amino acid sequence represented by SEQ ID NO:7 or
 8. 40.The cell according to any one of claims 1 to 28, which is capable ofdifferentiating into a cardiomyocyte or a blood vessel bytransplantation into a heart.
 41. The cell according to any one ofclaims 1 to 19, which is capable of differentiating into a cardiacmuscle by transplantation into a blastocyst or by co-culturing with acardiomyocyte.
 42. The cell according to any one of claims 1 to 28,which is capable of differentiating into an adipocyte by an activator ofa nuclear receptor, PPAR-Y.
 43. The cell according to claim 42, whereinthe activator is a compound having a thiazolidione skeleton.
 44. Thecell according to claim 43, wherein the compound is at least oneselected from the group consisting of troglitazone, pioglitazone, androsiglitazone.
 45. The cell according to any one of claims 1 to 28,which is capable of differentiating into a nervous cell bytransplantation into a blastocyst or by transplantation into anencephalon or a spinal cord.
 46. The cell according to any one of claims1 to 28, which is capable of differentiating into a hepatic cell bytransplantation into a blastocyst or by transplantation into a liver.47. A method for differentiating the cell according to any one of claims1 to 28 into a cardiac muscle, comprising using a chromosomalDNA-dimethylating agent.
 48. A method for redifferentiating the cellaccording to claim 9 into the cell according to 12, comprising using achromosomal DNA-dimethylating agent.
 49. A method for redifferentiatinga cell which is CD117-negative and CD140-positive into the cellaccording to claim 8, comprising using a chromosomal DNA-dimethylatingagent.
 50. The method according to claim 48 or 49, wherein thechromosomal DNA-dimethylating agent is selected from the groupconsisting of a demethylase, 5-azacytidine, and DMSO.
 51. The methodaccording to claim 50, wherein the demethylase comprises the amino acidsequence represented by SEQ ID NO:1.
 52. A method for differentiatingthe cell according to any one of claims 1 to 28 into a cardiac muscle,comprising using a factor which is expressed in a cardiogenesis regionof a fetus or a factor which acts on differentiation into acardiomyocyte in a cardiogenesis stage of a fetus.
 53. The methodaccording to claim 52, wherein the factor which is expressed in acardiogenesis region of a fetus or the factor which acts ondifferentiation into a cardiomyocyte in a cardiogenesis stage of a fetusis at least one selected from the group consisting of a cytokine, anadhesion molecule, a vitamin, a transcription factor, and anextracellular matrix.
 54. The method according to claim 53, wherein thecytokine is at least one selected from the group consisting of aplatelet-derived growth factor, PDGF; a fibroblast growth factor-8,FGF-8; an endothelin 1, ET1; a midkine; and a bone morphogenetic factor,BMP-4.
 55. The method according to claim 54, wherein the PDGF, FGF-8,ET1, midkine, and BMP-4 comprise the amino acid sequence represented bySEQ ID NO:3 or 5, the amino acid sequence represented by SEQ ID NO:64,the amino acid sequence represented by SEQ ID NO:66, the amino acidsequence represented by SEQ ID NO:68, and the amino acid sequencerepresented by SEQ ID NO:70, respectively.
 56. The method according toclaim 53, wherein the adhesion molecule is at least one selected fromthe group consisting of a gelatin, a laminin, a collagen, and afibronectin.
 57. The method according to claim 53, wherein the vitaminis retinoic acid.
 58. The method according to claim 53, wherein thetranscription factor is at least one selected from the group consistingof Nkx2.5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND,TEF-1, TEF-3, TEF-5, and MesP1.
 59. The method according to claim 58,wherein the Nkx2.5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND,eHAND, TEF-1, TEF-3, TEF-5, and MesP1 comprise the amino acid sequencerepresented by SEQ ID NO:9, the amino acid sequence represented by SEQID NO:11, the amino acid sequence represented by SEQ ID NO:13, the aminoacid sequence represented by SEQ ID NO:15, the amino acid sequencerepresented by SEQ ID NO:17, the amino acid sequence represented by SEQID NO:19, the amino acid sequence represented by SEQ ID NO:21, the aminoacid sequence represented by SEQ ID NO:23, the amino acid sequencerepresented by SEQ ID NO:25, the amino acid sequence represented by SEQID NO:27, the amino acid sequence represented by SEQ ID NO:29, the aminoacid sequence represented by SEQ ID NO:62, respectively.
 60. The methodaccording to claim 53, wherein the extracellular matrix is anextracellular matrix derived from a cardiomyocyte.
 61. A method fordifferentiating the cell according to any one of claims 1 to 28 into anadipocyte, comprising using an activator of a nuclear receptor, PPAR-γ.62. The method according to claim 61, wherein the activator is acompound having a thiazolidione skeleton.
 63. The method according toclaim 62, wherein the compound is at least one selected from the groupconsisting of troglitazone, pioglitazone, and rosiglitazone.
 64. Amyocardium-forming agent, comprising, as an active ingredient, achromosomal DNA-demethylating agent.
 65. The myocardium-forming agentaccording to claim 64, wherein the chromosomal DNA-demethylating agentis at least one selected from the group consisting of a demethylase,5-azacytidine, and DMSO.
 66. The myocardium-forming agent according toclaim 65, wherein the demethylase comprises the amino acid sequencerepresented by SEQ ID NO:1.
 67. A myocardium-forming agent, comprising,as an active ingredient, a factor which is expressed in a cardiogenesisregion of a fetus or a factor which acts on differentiation into acardiomyocyte in a cardiogenesis stage of a fetus.
 68. Themyocardium-forming agent according to claim 67, wherein the factor whichis expressed in a cardiogenesis region of a fetus or the factor whichacts on differentiation into a cardiomyocyte in a cardiogenesis stage ofa fetus is at least one selected from the group consisting of acytokine, an adhesion molecule, a vitamin, a transcription factor, andan extracellular matrix.
 69. The myocardium-forming agent according toclaim 68, wherein the cytokine is at least one selected from the groupconsisting of a platelet-derived growth factor, PDGF; a fibroblastgrowth factor-8, FGF-8; an endothelin 1, ETI; a midkine; and a bonemorphogenetic factor, BMP-4.
 70. The myocardium-forming agent accordingto claim 69, wherein the PDGF, FGF-8, ET1, midkine, and BMP-4 comprisethe amino acid sequence represented by SEQ ID NO:3 or 5, the amino acidsequence represented by SEQ ID NO:64, the amino acid sequencerepresented by SEQ ID NO: 66, the amino acid sequence represented by SEQID NO:68, and the amino acid sequence represented by SEQ ID NO:70,respectively.
 71. The myocardium-forming agent according to claim 68,wherein the adhesion molecule is selected from the group consisting of agelatin, a laminin, a collagen, and a fibronectin.
 72. Themyocardium-forming agent according to claim 71, wherein the vitamin isretinoic acid.
 73. The myocardium-forming agent according to claim 68,wherein the transcription factor is at least one selected from the groupconsisting of Nkx2.5/Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND,eHAND, TEF-1, TEF-3, TEF-5, and MesP1.
 74. The myocardium-forming agentaccording to claim 73, wherein the Nkx2.5/Csx, GATA4, MEF-2A, MEF-2B,MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5, and MesP1 comprisethe amino acid sequence represented by SEQ ID NO:9, the amino acidsequence represented by SEQ ID NO:11, the amino acid sequencerepresented by SEQ ID NO:13, the amino acid sequence represented by SEQID NO:15, the amino acid sequence represented by SEQ ID NO:17, the aminoacid sequence represented by SEQ ID NO:19, the amino acid sequencerepresented by SEQ ID NO:21, the amino acid sequence represented by SEQID NO:23, the amino acid sequence represented by SEQ ID NO:25, the aminoacid sequence represented by SEQ ID NO:27, the amino acid sequencerepresented by SEQ ID NO:29, and the amino acid sequence represented bySEQ ID NO:62, respectively.
 75. The myocardium-forming agent accordingto claim 68, wherein the extracellular matrix is an extracellular matrixderived from a cardiomyocyte.
 76. A method for regenerating a heartdamaged by a heart disease, comprising using the cell according to anyone of claims 1 to
 46. 77. An agent for cardiac regeneration,comprising, as an active ingredient, the cell according to any one ofclaims 1 to
 46. 78. A method for specifically transfecting a wild-typegene corresponding to a mutant gene in a congenital genetic disease to amyocardium, comprising using the cell according to any one of claims 1to 46 into which the wild-type gene corresponding to a mutant gene in acongenital genetic disease of a heart has been introduced.
 79. Atherapeutic agent for a heart disease, comprising, as an activeingredient, the cell according to any one of claims 1 to 46 into which awild-type gene corresponding to a mutant gene in a congenital geneticdisease of a heart has been introduced.
 80. A method for producing anantibody which specifically recognizes the cell according to any one ofclaims 1 to 46, comprising using the cell as an antigen.
 81. A methodfor isolating a cell having the potential to differentiate into acardiomyocyte according to any one of claims 1 to 46, comprising usingan antibody obtained by the method according to claim
 80. 82. A methodfor obtaining a surface antigen specific for the cell according to anyone of claims 1 to 46, comprising using the cell.
 83. A method forscreening a factor which proliferates the cell according to any one ofclaims 1 to 46, comprising using the cell.
 84. A method for screening afactor which induces the cell according to any one of claims 1 to 46 todifferentiate into a cardiomyocyte, comprising using the cell.
 85. Amethod for screening a factor which immortalizes the cell according toany one of claims 1 to 46, comprising using the cell.
 86. A method forimmortalizing the cell according to any one of claims 1 to 46,comprising expressing a telomerase in the cell.
 87. The method accordingto claim 86, wherein the telomerase comprises the amino acid sequencerepresented by SEQ ID NO:31.
 88. A therapeutic agent for a heartdisease, comprising, as an active ingredient, the cell according to anyone of claims 1 to 46 which has been immortalized by expressing atelomerase.
 89. The therapeutic agent according to claim 88, wherein thetelomerase comprises the amino acid sequence represented by SEQ IDNO:31.
 90. A culture supernatant comprising the cell according to anyone of claims 1 to
 46. 91. A method for inducing the cell according toany one of claims 1 to 46 to differentiate into a cardiomyocyte,comprising using the culture supernatant according to claim 90.